Accepted Manuscript

Terahertz biophotonics as a tool for studies of dielectric and spectral properties of

biological tissues and liquids

O.A. Smolyanskaya, N.V. Chernomyrdin, A.A. Konovko, K.I. Zaytsev, I.A. Ozheredov,
O.P. Cherkasova, M.M. Nazarov, J.-P. Guillet, S.A. Kozlov, Yu.V. Kistenev, J.-L.
Coutaz, P. Mounaix, V.L. Vaks, J.-H. Son, H. Cheon, V.P. Wallace, Yu. Feldman, I.
Popov, A.N. Yaroslavsky, A.P. Shkurinov, V.V. Tuchin

PII: S0079-6727(18)30045-4
DOI: https://doi.org/10.1016/j.pquantelec.2018.10.001
Reference: JPQE 219

To appear in: Progress in Quantum Electronics

Please cite this article as: O.A. Smolyanskaya, N.V. Chernomyrdin, A.A. Konovko, K.I. Zaytsev, I.A.
Ozheredov, O.P. Cherkasova, M.M. Nazarov, J.-P. Guillet, S.A. Kozlov, Y.V. Kistenev, J.-L. Coutaz,
P. Mounaix, V.L. Vaks, J.-H. Son, H. Cheon, V.P. Wallace, Y. Feldman, I. Popov, A.N. Yaroslavsky,
A.P. Shkurinov, V.V. Tuchin, Terahertz biophotonics as a tool for studies of dielectric and spectral
properties of biological tissues and liquids, Progress in Quantum Electronics, https://doi.org/10.1016/
j.pquantelec.2018.10.001.

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 ACCEPTED MANUSCRIPT

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Terahertz biophotonics as a tool for studies of
dielectric and spectral properties of biological

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tissues and liquids

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O.A. Smolyanskaya*1 , N.V. Chernomyrdin2,3,4 , A.A. Konovko5 , K.I. Zaytsev*2,3,4 ,
I.A. Ozheredov5 , O.P. Cherkasova4,6,7 , M.M. Nazarov8 , J.-P. Guillet9 , S.A. Kozlov1 ,
Yu.V. Kistenev7 , J.-L. Coutaz10 , P. Mounaix9 , V.L. Vaks11 , J.-H. Son12 , H. Cheon12 ,
V.P. Wallace13 , Yu. Feldman14 , I. Popov14 , A.N. Yaroslavsky15 , A.P. Shkurinov*5,18,19 ,

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and V.V. Tuchin1,7,16,17

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1
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ITMO University, Saint-Petersburg 199004, Russia
Bauman Moscow State Technical University, Moscow 105005, Russia
Sechenov First Moscow State Medical University, Moscow 119991, Russia
4
Prokhorov General Physics Institute of the Russian Academy of Sciences, Moscow
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119991, Russia
5
Department of Physics and International Laser Center, Lomonosov Moscow State
University, Moscow 119991, Russia
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Institute of Laser Physics of SB RAS, Novosibirsk 630090, Russia
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7
Tomsk State University, Tomsk 634050, Russia
8
National Research Center "Kurchatov Institute", Moscow 123182, Russia
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9
IMS Laboratory, UMR CNRS 5218, University of Bordeaux, Talence 33405, France
10
IMEP-LAHC, UMR 5130 CNRS, University Savoie Mont Blanc, Le Bourget du Lac
73376, France
11
Institute for Physics of Microstructures of RAS, N. Novgorod 603087, Russia
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12
Department of Physics, University of Seoul, Seoul 02504, South Korea
13
Department of Physics, The University of Western Australia, Perth, 6009, Australia
14
Department of Applied Physics, Edmond J. Safra Campus, Givat Ram, The Hebrew
University of Jerusalem, Jerusalem 91904, Israel
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15
Advanced Biophotonics Laboratory, University of Massachusetts, Lowell 01854, USA
16
Saratov State University, Saratov 410012, Russia
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Institute of Precision Mechanics and Control of RAS, Saratov 410028, Russia
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The National University of Science and Technology MISiS, Moscow 119049,Russia
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Institute on Laser and Information Technologies of RAS, Branch of the FSRC
“Crystallography and Photonics,” RAS, Shatura, Moscow Region 140700, Russia

November 2, 2018

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Abstract
In this review, we describe dielectric properties of biological tis-

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sues and liquids in the context of terahertz (THz) biophotonics. We
discuss a model of the THz dielectric permittivity of water and water-
containing media, which yields analysis of the relaxation and damped
resonant molecules modes. We briefly describe modern techniques

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of THz spectroscopy and imaging employed in biophotonics with a
strong emphasize on a THz time-domain spectroscopy. Furthermore,
we consider the methods of sub-wavelength resolution THz imaging

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and the problem of THz wave delivery to hard to access tissues and in-
ternal organs. We consider the THz dielectric properties of biological
solutions and liquids. Although strong absorption by water molecules
prevents THz-waves from penetration of hydrated tissues and prob-

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ing biological molecules in aqueous solutions, we discuss approaches
for overcoming these drawbacks – novel techniques of freezing and
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temporal dehydration by application of hyperosmotic agents which
have a potential for cancer detection. We review recent applications
of THz technology in diagnosis of malignancies and aiding histology
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paying particular attention to the origin of contrast observed between
healthy and pathological tissues. We consider recent applications of
THz reflectometry in sensing the thinning dynamics of human pre-
corneal tear film. Modern modalities of THz imaging, which relies
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on the concepts of multi-spectral and multi-temporal domains and

employing the principles of color vision, phase analysis and tomogra-
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phy are discussed. Novel methods of THz spectra analysis based on

machine learning, pattern recognition, chemical imaging and the re-
vealing of the spatial distribution of various substances in a tissue, are
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analyzed. Advanced thermal model describing biological object irra-

diated by THz waves and phantoms mimicking the optical properties
of tissues at THz frequencies are presented. Finally, application of the
high-resolution THz spectroscopy in analytic chemistry, biology and
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medicine are described.

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Keywords: Terahertz technology; terahertz biophotonics; terahertz spec-

troscopy and imaging; water and water solutions; biological tissues and liq-
uids; dielectric permittivity; sub-wavelength spatial resolution; diagnosis of
malignancies; tissue phantoms; optical clearing of tissues; terahertz-wave
penetration enhancement; terahertz waveguides; terahertz data analysis and
processing

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1 Introduction
Terahertz (THz) science and technology have been challenging but excit-

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ing areas of research for the last years. As THz radiation lies between mi-
crowave and optical ranges, it demonstrates the features of both of them. In
the 1980s, when active development of laser-based THz technologies started,

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one of the applications that appeared then was their use in the medico-
biological sphere. High informativeness of THz radiation is explained by
the specific response of complex molecules of biological systems. Since the

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very beginning, it has been clear that the main informative feature and si-
multaneously the main obstacle is water. Its intensive absorption in this
frequency range, on the one hand, limits its penetration into biological tis-
sues, but, on the other hand, allows one to consider it as the subject of a

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separate fruitful study. The energy of hydrogen bonds, which are the basis
of all biological systems, is comparable with the energy of ”THz quantum”.
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Therefore, a water molecule itself can be considered as a universal marker
of THz frequency range, which is sensitive to various vital processes going
on in living tissues and cells. In comparison with what is traditionally de-
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scribed in dielectric spectroscopy, in THz frequency range, water as a marker
enables us to obtain new information about biological systems. Moving from
gigahertz (GHz) to THz range, we gradually approach various oscillation
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processes, which are determined by the interaction of water molecules with

the surrounding molecular systems.
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Water molecules can be a structural unit of a large enzyme molecule or a

solvent, in other words, can be free or bound. Water is considered to be the
most important substance in tissues of our body [1]. Since P. Debye intro-
duced the rotational diffusion model of the liquid in 1929, many researchers
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have studied the dielectric response of the water. In Fig. 1, we show results of
the broadband spectroscopy of bulk water reprinted from the Ref. [2], where
the red and black curves stand for the absorption coefficient α and the index
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of refraction n, respectively, while the gray and yellow areas indicate the THz
and visible parts of the spectrum. Most researchers identified two relaxation
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modes of the water: one is located at the range of GHz to 0.1 THz, called
as a slow relaxation water at the time scale of ' 10 ps. Another is start-
ing to be seen at the region of THz, called as a fast relaxation water at the
time scale of sub-picoseconds. Do these relaxation times reflect any micro-
scopic relevancies? Previous dielectric spectroscopy studies have proposed
that the origin of the fast relaxation water is linked with water’s hydrogen

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bond networks. However, the molecular-level mechanisms controlling this

fast relaxation behavior remains essentially unknown.
All biological tissues contain water in amount from a few percent of their

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weight for fat and some hard tissues, like teeth, and up to 85 % for soft tissues,
like lungs, and tendon of newborns. For humans, the total body water in
norm is about 68 %, the skin contains 64 % of water, muscles and kidneys

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– 79 %, the brain and heart – 73 %, lungs – 83 %, bones – 31 %, and only
teeth are dry enough with 5 % of water [3]. Understanding the properties
of tissue water in THz range is fundamental because of strong absorption of

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radiation and the sensitivity to metabolic hydration of malignant neoplasms
of human tissues [2, 4–7].
Biological tissues contain water in two main states – bound and free.
Bound water is strongly associated with tissue molecular components and

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is not movable, except strong tissue compression [8] or heating [9]. Free
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water is a major component of tissue water [7]; it presents in interstitial
fluid and cell cytoplasm and can move from one space to another or even
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Figure 1: Results of the broadband spectroscopy of bulk water: red curve

stands for the absorption spectrum, and black curve stands for the refractive
index. The gray area indicates the 0.1–2.5 THz region, and the narrow yellow
shaded area indicates the visible part of the spectrum. (Reprinted from the
Ref. [2], Optical Society of America)

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outside, being stimulated by osmotic or mechanical pressure. When the

appropriate (strong) stimulation is applied to the tissue, the bound water
can be converted into free one and transferred to the outside [8–10]. The

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inverse pathway is also possible depending on the experimental condition.
The exchange of water between the bound and free states and between the
free and the environmental state can be described by the following equations

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for water concentrations Ni (i = free, env, bound) [10]:
dNfree
= α1 (Nenv − Nfree ) + α2 (Nbound − Nfree ) , (1)

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dt
dNbound
= α2 (Nfree − Nbound ) , (2)
dt

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where α1 represents the exchange rate between the environment and the free
state, while α2 represents the exchange rate between the free and bound
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states. As an example, in Fig. 2, the THz properties of thin slices of sheep
liver tissue, extracted from the reflection-mode spectroscopy measurements,
are presented. These data clearly reveal high sensitivity of THz waves to
the water content and its state in tissue. Useful information here is in the
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difference between bulk water spectra and those ones with bound water.
For precise values of water absorption and refraction see below in section
2.1.2. A more accurate and detailed gradation of bound water is described
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in Refs. [11, 12].

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(a) (b)
Figure 2: THz characteristic properties of bulk water, bulk water plus bound
water, and sheep liver tissue: (a) absorption coefficient; (b) refractive index.
(Reprinted from the Ref. [4])

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The paper is organized as the following.

In section 2, we describe a number of models of a water dielectric per-
mittivity, which are extended to the THz frequency range. These models

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consider both frequency and temporal responses of the water containing me-
dia, which also allow one to analyze isolated molecular resonances, even if
they are damped. By combining the models of dielectric media with the

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models of effective media described in this paper, we can analyze complex
biological tissues.
In section 3, the most popular techniques of investigation in the far-

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infrared, namely the time-domain spectroscopy (TDS) and the Fourier-transform
infrared spectroscopy (FTIR), are described and compared in terms of per-
formance with a strong emphasize on a detailed description of TDS. Also the
complementary technique such as Raman spectroscopy is presented.

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In section 4, we show that since the THz range is located between optics
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and microwaves, many radiation sources, detectors and optical components
are designed using different existing principles. We discuss recent advances in
electronic technologies that can be applied in biology and medicine. Further-
more, we discuss modern modalities of THz imaging, which allow for bringing
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the spatial resolution to essentially subwavelength scales and, thus, for visual-
ization the sub-wavelength features of tissues (i.e., separate cells, microfibrils
and cell organelles), as well as prospects of their use in THz biomedicine.
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Finally, we summarize recent developments in the area of THz waveguides

and endoscopes, which are based on various materials and exploit different
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physical principles for the THz radiation delivery to hardly-accessible biolog-

ical tissues and internal organs; thus, significantly expending possibilities of
THz medical diagnosis.
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In section 5, we give a detailed description of biological solutions and

bioliquids (protein and sugar solutions, blood components). We analyze the
most advanced measuring techniques and present recently proposed approach
to the description of the dielectric function of biological solutions in the THz
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range. High THz-wave absorption by water helps to distinguish the cancer-

ous tissue among the normal one by exploiting such marker as a difference of
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water content. Although strong absorption prevents deep THz-wave penetra-

tion into hydrated tissues and correspondingly probing of biological molecules
in aqueous solutions. This drawback can be overcome by the novel freezing
technique which is applicable in cancer detection.
In section 6, we consider application of THz spectroscopy and imaging in
noninvasive, least-invasive and intraoperative diagnosis of malignant tissues

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in various localizations of human body, as well as in aiding histology of tissue

specimens. We discuss a typical character of the dielectric response of tissues
at THz frequencies and comment on the origin of contrast observed between

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normal and malignant tissues in THz spectra and images.
In section 7, we demonstrate the applicability of the THz reflectometry
technique for experimental study of human eye cornea hydration. We show

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the possibility of determination of the dielectric permittivity of an eye cornea
sample for its different hydration levels. The proposed procedure based on
normalization of the measured cornea sample permittivity to the parameters

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of nanostructured aluminum oxyhydroxide is described. Frequency-domain
THz reflectometry approach suggested for in vivo sensing of thinning dynam-
ics of human precorneal tear film is presented.
In section 8, we analyze a development of the THz imaging approach,

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based on the concepts of multispectral and multi-temporal domains, which
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combine the principles of color vision, phase analysis and tomography in THz
image acquisition. We also present the application of the proposed technique
for studies of biological tissues, illustrated by the human skin cancer detec-
tion.
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In section 9, we discuss methods of THz spectrum analysis based on
machine learning, including pattern recognition approaches. We describe
modern methods of THz chemical imaging revealing spatial distribution of
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various molecular substances in a biological tissue. However, identification

of pure compounds using molecular signatures in the THz range is still not
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straightforward because of the inherently broad spectral signatures in liquids

and tissues.
In sections 10-12, we demonstrate a new thermal model of biological
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object irradiated by pulsed or continuous-wave (CW) THz radiation (sec-

tion 10), an approach for increasing the depth of THz wave penetration
into tissues by temporal and reversible tissue dehydration (section 11), and
novel phantoms mimicking the optical properties of tissues in the THz range
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(section 12). Tissue phantoms designed to provide periodic calibration, val-

idation, and quality assurance for THz devices are described.
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In section 13, we consider instruments and applications of the high-

resolution THz spectroscopy, which is capable for solving numerous demand-
ing problems in analytical chemistry, biology and medicine.

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2 Models of terahertz radiation interaction

with liquids and tissues

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2.1 Water and dielectric function
2.1.1 Background of terahertz spectroscopy of water solutions

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It is generally accepted that liquid water plays a dominant role in bi-
ology; water molecules form hydrogen bonds (HB) with their neighbors to

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construct the water network with a, suggested, tetrahedral-like structure.
Evidently, both the high density of hydrogen bonding sites and the equal
number of proton donors and acceptors renders water unique among all liq-
uids. Some features of the water molecule, however, become apparent to

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mainly cause the outstanding properties of water as liquid and solvent [13].
Of particular importance is the interaction of water molecules with dissolved
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biological molecules. Currently, water can be divided on bulk water (it does
not form bonds with biomolecules) and hydration or bound water (it sur-
rounds biomolecules and interacts with them) [14, 15]. Thus, in the solution,
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a considerable part of water molecules are in the form of a hydration shell.
THz-TDS fortunately covers the spectral range of HB response [16], al-
though strong absorption makes its measurement challenging. All known
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processes in polar solutions below the tens of THz have very broadband re-
sponses, so it is essential to combine several experimental techniques, each
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for its own spectral range, to obtain spectra over many octaves. From the
low frequency side, it is well established dielectric spectroscopy [17]; from the
high frequency side, it is mostly FTIR [18]. In paper [19], the THz dielec-
tric response is systematically studied and modeled in the frequency range
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of several GHz to 25 THz and in the temperature range of 0 to 100 o C.

The specific feature of THz spectroscopy of solutions is a very strong
absorption and dispersion by the solvent (polar liquid) itself. Actually, we
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do not measure the solute, its contribution is negligible, we measure how the
solute modifies the solvent, the appearance and the structure of hydration
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shell around the solute molecules. To detect small changes in the solution
smooth spectra, we should precisely describe solvent properties in a broad
spectral range.

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2.1.2 The relaxation models of the bulk water

To fit dielectric function ε(ω) spectrum of a water a well-known model

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with Debye-type relaxation (εrelax ) and over-dumped oscillator (εosc ) compo-
nents is usually applied [13].
Generally, for THz frequency range the function is

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ε(ω) = ε∞ + εrelax + εosc + εcond , (3)
where ω = 2πν is the angular frequency of the external electric field; ε∞ = n2

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stands for the high frequency limit of complex dielectric permittivity; the last
term εcond = σ/(jωε0 ) describes the losses due to the ionic conductivity σ,
where ε0 is the permittivity of a free space [20]. There is an important
condition on the amplitudes of considered terms in Eq. (3)

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Ai
εs = ε∞ + ∆εm + (4)
X X
ANm i ω0,i 2
,

where εs is the precisely-measured low-frequency limit of the dielectric per-

mittivity, ∆εm is the amplitude of relaxation process m; Ai /ω0,i2
is the ampli-
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tude normalized to corresponding frequency ω0,i of the overdumped oscillator
i. From Eq. (4), it follows that ε∞ value depends on the number of consid-
ered processes m and i, which is also illustrated in Fig. 3 (a) and by the data
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presented in Tab. 1.
The physical processes behind terms in Eq. (3) are the following.
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The relaxation process εrelax ∼ ∆ε/ (1 + jωτ ) reflects the cooperative

reorientational dynamics of the dipole moment. This is interpreted as either
a co-operative process or one involving making and breaking of the hydrogen
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bonds; τ is assigned to the cooperative reorientation time of hydrogen-bond

structure of bulk water molecules involving HB switching events [22, 23].
The oscillation processes εosc ∼ A/ (ω02 − ω 2 + jγ0 ω) indicates the over-
damped modes, which correspond to several known vibration modes in the
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THz region, typically those frequencies are expressed in wavenumbers: η[cm−1 ] =

ν[THz] · 33.3. The lowest resonance is around η = 50-60 cm−1 , that is
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ν = 1.5 THz the bending mode between two water molecules forming the
hydrogen-bond; it is temperature-independent and strongly suppressed in
the infrared spectra, corresponding to maximum 3% in the imaginary part
of pure water and, thus, is commonly neglected [22]. At the frequency of
η = 180 cm−1 (ν = 5.4 THz), the intermolecular stretching of water is as-
signed to a hindered O...O translation. The intermolecular libration at the

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Figure 3: The complex dielectric spectrum of water; the blue symbols rep-
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resent the residual dielectric loss after subtracting the contribution of the τ1
process (a). Raman spectrum of water (b). (Reprinted from the Ref. [21],
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copyright (2018) by the American Physical Society)

frequency of η = 420 cm−1 (ν = 12 THz) of water is assigned to a hindered

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O...H rotation [21, 22]. This and higher frequency processes have negligible
contributions below 3 THz and, therefore, we will skip them.
The width of spectral response of all overlapped processes is so broad
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that the central frequency (or the relaxation time) is determined with a
large error; moreover, replacement of the relaxation process by a resonant
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one preserves an acceptable accuracy of the description of the real spectrum.

All the mentioned processes except the slow relaxation are also pronounced
in Raman spectroscopy; see Fig. 3.
The only not-completely explained process is the so-called ”fast relax-
ation” (m = 2). In fact, there is some additional wing to the slow relaxation
process, a significant contribution of which manifests itself between 200 GHz

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and 3 THz – just the range of the optimum sensitivity of the THz-TDS.
With the accuracy that is available in modern experiments, the introduction
of the ”fast relaxation” term describes the measured spectra well. However,

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the absolute values of this relaxation time and the amplitude depend on the
frequency range where the optimal fit is carried out; see Tab. 1.

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 Table 1: Parameters of the dielectric function of water.
Here, TDS stands for the transmission-mode THz time-domain spectroscopy; ATR stands for the attenuated
total internal reflection THz time-domain spectroscopy; DS stands for dielectric spectroscopy; FTIR stands
for Fourier-transform infrared spectroscopy; Raman stands for the Raman (or combinational scattering)
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spectroscopy. (Courtesy of M.M. Nazarov)
C 2
Frequency range, THz εs ε∞ ∆ε1 τ1 , ps ∆ε2 τ2 , fs ω0X , THz As /ω0S ω0S , THz γ0S , THz T, ◦ C Method Refs
3· 10−5− 0.1 78.4 5.2 73.16 8.27 0 - - 0 - - 25 DS [17]
0.08-2.7 77.5 2.45 73 9.5 1.7 360 - 1.12 5.3 5.35 22 TDS & ATR [24]
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0.1-2.0 78.8 3.20 73.6 8.0 1.60 180 - 0 - - 22 TDS [25]
0.2-13.5 79.9 2.00 74.90 9.43 1.67 248 - 1.12 5.3 5.35 20 ATR+IR [26]
0.25-4.0 0.01-0.04 77.8 2.39 72.02 7.93 2.1 260 - 1.27 5.22 5.68 27 DS & ATR [22]
1.5-6.67 78 2.20 71.49 8.311 2.80 and 1.6 1000 and 100 - 1.12 5.26 4.34 25 FTIR [18]
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0.18-2.42 4.54-6.06 78.4 - - 8.25 1.4 ± 0.1 310 ± 60 1.5 1.57 5.4 - 25 TDS & FTIR [20]
0.03-18 - - - 8.32 - 420 1.5 - 5.4 - 25 Raman & FTIR [21]
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All the parameter sets presented in Tab. 1 describe water dielectric func-
tion, absorption and refraction with a high precision in the given frequency
range.

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Figure 4: Models (lines) and experimental data (circles) of THz characteristic
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of water at room temperature: absorption coefficient (a) and refractive index
(b) spectra. (Courtesy of M.M. Nazarov)

Note, that it is important to fix the temperature, because with a temper-

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ature change, τslow significantly changes [19], which has a noticeable effect in
the low-frequency part of the THz spectrum.
It is worth mentioning that from a formal point of view the Debye-type
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unit in water permittivity is the degenerate case of more general theoretical

approach developed by H. Fröhlich [27]. He suggested to introduce formally
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relaxation time distribution function f (τ ) instead of one single relaxation

time. This approach means that there is reorientation energy barrier dis-
tribution in the dipole molecule media. Thus Debye-like equation for ε in
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Fröhlich’s form is
Z∞
f (τ )dτ
εrelax (ω) = . (5)
1 + jωτ
0
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In practice, one uses two types of function f (τ ), namely discrete and

continuous distributions. For example, the discrete distribution function
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f (τ ) = ∆εi δ (t − τi ) (6)
X

leads to Debye-like equation for water permittivity

∆εi
εrelax (ω) = (7)
X
.
i 1 + jωτi

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Every unit in the sum means the i-th process contribution to complex
permittivity, where τi is the relaxation time of i-th process; ∆εi is the con-
tribution of i-th process in permittivity ε; τ1 = τslow is the classical Debye

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relaxation time. Such a formal separation of relaxation behaviour into differ-
ent Debye-like relaxation processes occurs very useful for theoretical descrip-
tion of spectrum asymmetry at comparatively low frequency range. Namely,

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one additional unit describes a so-called ”fast” Debye relaxation with time
constant τ2 [26]
∆ε1 ∆ε2
εrelax (ω) = + (8)

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,
1 + jωτ1 1 + jωτ2
where τ1 corresponds to classical Debye relaxation time; τ2 corresponds to
fast Debye relaxation process; ∆ε1,2 means relative contribution of those

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processes to permittivity spectrum. The double-Debye permittivity appears
useful for description of tissue dielectric response at THz frequencies as will
be shown below. AN
Recently, another approach of the dielectric relaxation of bulk water based
on the defect migration model was developed [28]. This model does not
require the additional “fast” Debye process as in the two-fraction water model
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to describe the excess wing in the sub THz region. In this study, the “wait-
and-switch” model was used as a basic model of water relaxation model
[13, 29, 30]. According to this model, the reorientation of a water molecule
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occurs on the large angle and only when it encounters an appropriate defect
of the H-bond network, otherwise the water molecule remains in a waiting
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mode. This waiting regime leads to a delay in the relaxation compared with
the gaseous state, where the relaxation time is around 1 ps [31]. In other
words, the change of the total polarization is caused by the migration of the
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defects through the H-bond network.

Under the term of “defects” it is implied the orientation (or structural)
defects, which present any irregularities of the ideal tetrahedral ordering of
the H-bond network in bulk water (see Fig. 5 (a)). For instance, it can be
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bifurcated (see Figs. 5 (b) and (c)), trifurcated H-bonds, or has other more
complicated configurations [32]. The presence of any defect in the vicinity of
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the water molecule facilitates its reorientation. For example, the bifurcated
hydrogen bond lowers the potential energy barrier for reorientation of the
fivefold H-bonded water molecule (see Fig. 5 (d)). Hence, the fifth neighbor
supports reorientation motion by the simultaneous action of two favorable
effects: weakening of existing hydrogen bonds and provision of an appropriate

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Figure 5: Orientation defects: Local tetrahedral ordering of the water

molecules without defects (a). The defect of the H-bond network due to
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bifurcated hydrogen (b). The defect of the H-bond network due to bifur-
cated oxygen (c). Schematic representation of the migration of orientation
defects (pink shaded area) with consecutive changing of the dipole moment’s
direction of water molecules (d). (Reprinted from the Ref. [28], reproduced
C

by permission of the PCCP Owner Societies)

AC

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site for the formation of a new hydrogen bond. Further migration of the
orientation defects leads to consecutive reorientation of the dipole moment’s
direction of water molecules (see Fig. 5 (d)). Furthermore, by implying

PT
the switching reorientation of water molecules, we may then suggest the
hopping of orientation defects between molecules rather than their continuous
diffusion. This mechanism is described in detail in Refs [13, 32–35] and is

RI
supported by various experimental observations [30, 34, 36]. In a way, the
orientation defects in water resemble the L-D Bjerrum defects in ice [37].
However, the defects in water are smeared out and not localized [32].

SC
It is worth to note that in addition to the orientation defects, the polar-
ization of water may be caused by migration of H3 O+ and OH pairs, also
known as ionic defects [38]. However, the strong temperature correlation of
the dielectric time relaxation, τD , with viscosity, η [39], proton spin-lattice

U
relaxation time, T1 [40], [41], and the absence of the reaction of the dielectric
AN
relaxation upon changes in pH [42–45], leads us to the assumption that mi-
gration of the orientation defects should be considered as a main mechanism
of the dielectric relaxation in water.
In the paper [28], the idea of orientation defects migration was signifi-
M
cantly developed and the relationship between complex dielectric permittiv-
ity and the mean square displacement (MSD) of the defects was derived
D

∆ε
ε∗ (ω) = ε∞ + h i−1 . (9)
iωnq 2
1+ hr∗2 (ω)i
TE

6kB T ε0

Here q, n and hr∗2 (ω)i are the effective charge, number density and Fourier
transform of the mean square displacement of the defect, respectively; ε0 =
8.854 · 10−12 F/m is the permittivity of vacuum; and kB is the Boltzmann
EP

constant. In the case of normal diffusion behavior

D E
r2 (t) = 6Ddefect t, (10)
C

where Ddefect is the diffusion coefficient of the defect migration. Substituting

the Fourier transform of Eq. (10) hr∗2 (ω)i = 6Ddefect (iω)−2 into Eq.(9) yields
AC

the well-recognized Debye equation

∆ε k B T ε0
ε∗ (ω) = ε∞ + , τdefect = . (11)
1 + jωτdefect nq 2 Ddefect
Note that this equation provides the perfect description of the water dielectric
spectra only up to tens of GHz. In the similar study [28], based on the

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analysis of dielectric and Raman spectra, it was implied that the vibration of
the H-bond network starts to play an important role in the sub-THz range.
In terms of defect migration, it means that each defect has an additional

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vibrational motion near the water molecule before its consequent movement.
Thus, the whole defect movement can be expressed as a superposition of
translation rtr (t) and oscillation uosc (t) motions as follows

RI
D E D E D E
r2 (t) = rtr
2
(t) + u2osc (t) . (12)

SC
The translation term is responsible for the defect migration from one molecule
to another and can still be considered to be a normal diffusion hrtr 2
(t)i =
6Ddefect t [46]. Whereas, the oscillation term is shown to have the power law
behavior hu∗2osc (ω)i = (jωτosc )
−(1+δ)
, where δ is related to spectral dimension

U
of the H-bond network [47, 48]. Finally, the complex dielectric permittivity
is defined as follow

ε∗ (ω) = ε∞ +
AN
h
∆ε
i−1 . (13)
1 + (jωτdefect )−1 + (jωτosc )−δ
M
The function Eq. (13) has the usual Debye-type behavior at low frequencies,
while at high frequencies the excess wing appears with a slope equal to δ in
log-log scale. The asymmetrical shapes of the spectra produced by Eq. (13)
D

at different values of δ are presented in Fig. 6.

We see that the specific behavior of function Eq. (13) behind the maxi-
TE

mum of the losses peak allows us to describe the non-Debye behavior of the
water relaxation in the sub-THz region. In this case, we have no need to in-
troduce the additional “fast” Debye process as in two-fraction water model.
EP

Taking into account the additional terms corresponded to resonances at fre-

quencies ~60, 180, 400 and 700 cm−1 (which are observed in FIR [49,50] and
low-frequency Raman spectroscopy [21, 51] measurements), finally we obtain
the formula for fitting dielectric water spectra up to 10 THz
C

2
A ω60 2
A180 ω180
ε∗ (ω) = ε1,∞ + + ω2 −ω602 +jωΓ + +
AC

∆ε
1+[(jωτdefect ) +(jωτosc ) ]
−δ −1
−1 2 2
ω180 −ω +jωΓ180
60 60
2
A400 ω400 2
A700 ω700
+ ω2 −ω2 +jωΓ400 + ω2 −ω2 +jωΓ700 .
400 700
(14)
The fitting result for T=293K is presented in Fig. 7. The fitting parameters
of the first term in this case are equal to ∆ε = 75.3 ± 1.4, ε∞ = 1.7 ± 0.3,
τdefect = 12.4 ± 0.2 ps, δ = 0.93 ± 0.01, and τosc = 35 ± 1 ps, while the static

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permittivity is equal to εs = ∆ε+ε∞ = ∆ε+ε1,∞ +A50 +A180 +A400 +A700 ≈

80.
In addition to above mentioned theoretical models of permittivity, some

PT
empirical formulas are developed on the basis of continuous relaxation time
distribution functions. Among them, we consider the Cole-Cole model [52,53]
∆ε

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εrelax = , (15)
1 + (jωτα )α
where empirical parameter 0 6 α 6 1 describes the width of relaxation time

SC
spectrum; τα is the effective relaxation time. Another phenomenological
expression is the Davidson-Cole model [54]
∆ε
εrelax = , (16)

U
(1 + jωτM )β
where empirical parameter 0 6 β 6 1 and τM have similar physical sense.
AN
Notice, Eqs. (15) and (16) turn into the Debye model when assuming α = 1
M
D
TE
C EP
AC

Figure 6: Normalized model data of dielectric losses generated by Eq.(13)

at different values of δ. For each value of δ the spectrum is normalized
by the amplitude of the main dielectric peak and the frequency scale by
the characteristic of the same peak. Here we used the following parameter
values: ∆ε = 75, τdefect = 9 ps, τosc = 10 · τdefect . (Reprinted from the
Ref. [28], reproduced by permission of the PCCP Owner Societies)

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 ACCEPTED MANUSCRIPT

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RI
U SC
AN
M

Figure 7: Fitting of the dielectric spectrum of water at 20 o C: the black curve

D

is the fitting curve according to Eq. (14); the red curve represents the sum
of the first (ε1,∞ ) and the second terms of Eq. (14); the grey dashed curves
TE

describe the four last terms of Eq. (14); the blue curve represents Debye-
type behavior. (Reprinded from the Ref. [28], reproduced by permission of
the PCCP Owner Societies)
EP

and β = 1, respectively. Finally, the formal generalization of the Cole-Cole

and the Davidson-Cole models is given by the Havriliak-Negami function [55]
C

∆ε
εrelax = β. (17)
AC

(1 + (jωτα )α )

It is worth mentioning that the physical processes leading to the Davidson-

Cole, the Cole-Cole and the Havriliak-Negami formulas are not reliably known
[56].

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2.2 Dielectric model of aqueous protein solutions

Though generally biological samples are inhomogeneous, anisotropic and

PT
rough, the biological medium structural unit size is negligibly small compared
to the THz wavelengths in a number of important occasions (human red blood
cells, bovine serum albumin (BSA) etc.). In such cases, one can consider
such a biological sample as a homogeneous medium on the scale set by the

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THz wavelengths. Moreover, the spectra of aqueous protein solutions with
not extremely high concentration are almost completely determined by the
spectral properties of the solvent water [57]. Therefore, there are reasons for

SC
application of Eq. (3) to describe permittivity of such biological media as
aqueous protein solutions.
For example, according to the Ref. [57], the dielectric permittivity of the

U
aqueous phase of the solution in the presence of BSA ε(ω) is given by the
following expression

ε(ω) = ε∞ +
∆ε1
+
AN
∆ε2
+ 2
A1
+
jσ0
. (18)
1 − jωτ1 1 − jωτ2 ω1 − ω − jωγ1 ε0 ω
2
M
Using Eq. (18), the authors of Ref. [57] obtained six model parameters val-
ues calculated for solutions with and without BSA at the same pH. Thereby
the fitted solution permittivity model includes three water spectral bands:
D

two relaxation bands with relaxation times of about 8.28 and 0.30 ps and
a vibrational band with a maximum of about 180 cm−1 . The deviations of
TE

these parameters at different pH are significant. For instance the difference

in the mean values between the solutions with pH= 6 is < 0.52, whereas it
is > 3.10 for solutions with pH= 10. A comparison between the values of
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model parameters of solutions with BSA and without BSA suggests that the
main effect of BSA is the formation of strongly bound hydration shells in the
immediate proximity to the protein molecule. Various authors estimated the
hydration shells of proteins to be in the range of 10–50 Å [58–60], which cor-
C

responds to dozens of water molecule layers. At the same time, the structure
of more distant layers of the hydration shells is destroyed, resulting in the
AC

appearance of more free water molecules. Some variations for the effect of
different BSA conformation on the aqueous phase of solution are observed.
The proposed approach can be generalized and applied for studying of a wide
class of biological macromolecules in aqueous solutions [57].

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2.3 Complex biological medium permittivity based on

the effective medium theory

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Biological tissues are typically inhomogeneous and anisotropic, providing
strong scattering and extinction of the probing electromagnetic radiation. A
derivation of an analytical solution of the Maxwell’s equations for the elec-

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tromagnetic radiation for a biological sample interaction is quite a daunting
task, while a numerical solution of such problem is usually a very resource-
intensive and time-consuming problem. In this case, approximate models are

SC
of great importance. Nevertheless, on a number of important occasions, the
characteristic size of biological medium microinhomogeneity is much smaller
than THz wavelengths. This circumstance let us to consider such a medium
as homogeneous and, thus, to simplify this problem using an effective medium

U
model. The simplest effective media models are given by Maxwell Garnett,
Bruggeman and Landau-Lifshitz-Looyenga equations [61, 62].
AN
Maxwell Garnett model describes effective permittivity, εeff , of a two-
component medium. The first component is a homogeneous medium with a
permittivity ε2 , the second one is represented by spherical particles with a
M
permittivity ε1 . In this case, Maxwell Garnett equation is
εeff − ε2 ε1 − ε2
= f1 , (19)
εeff + 2ε2 ε1 + 2ε2
D

where f1 = (1/V ) i Vi is the filling factor, Vi is the volume of i-th particle;

P
TE

V is the total volume of the composite medium. It is assuming that spherical

particles are not clustered, and they are rather sparse.
The Bruggeman model is more symmetric with respect to different frac-
EP

tion permittivities. Moreover, this model is expanded easily to multicompo-

nent medium of spherical particles with different permittivities εi . In accor-
dance with the Bruggeman model, the effective medium permittivity is given
by:
C

N N
εi − εeff
= 0, fi = 1, (20)
X X
fi
εi + 2εeff
AC

i=1 i=1

where fi = (1/V ) j Vi,j is the filling factor of ith fraction. In the case of
P

two fractions, the model is often claimed to be valid under the following
restriction for the filling factors
1 2
<f < . (21)
3 3
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The Landau-Lifshitz-Looyenga model is given by an equation

D E
1/3
εeff = ε1/3 , (22)

PT
where angular brackets h. . .i denote volume averaging, and hεi is the average
permittivity.

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In addition to above mentioned models one can use the analytical repre-
sentation proposed by Bergman in the spirit of Fröhlich for porous composites

SC
Z1
 
g(x, p)dx
εeff = ε1 1 − (1 − p) (23)
ε1 / (ε1 − ε2 ) − x

0

where p is the porosity, g(p, x) is the geometrically deterministic function of

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the spectral density [63].
AN
Thereby the Bruggeman model is rather convenient for description of
multicomponent media. In the case of n-fraction medium, it leads to n-order
algebraic equation for εeff . One can choose the root taking into account the
sign of imaginary part of εeff . For example, the authors of the paper [57]
M
use the two-component Bruggeman model for description permittivity of a
protein aqueous solution
D

εp − εeff εw − εeff
fp + (1 − fp ) = 0, (24)
εp + 2εeff εw + 2εeff
TE

where fp and εp are the filling factor and permittivity of a protein, respec-
tively, εeff is the permittivity of protein aqueous solution and εw is the per-
mittivity of the aqueous phase of the solution in the presence of a protein.
EP

In the simplest case of two-component media, there are two roots of equa-
tion (20)

1
C

εeff,1,2 = [f1 (2ε1 − ε2 ) − f2 (ε1 − 2ε2 )] ±

4
1q
AC

± 8ε1 ε2 + (f1 (ε2 − 2ε1 ) + f2 (ε1 − 2ε2 ))2 . (25)

4
It is known that relaxation times of water molecules involved in hydration
of biomolecules are different from those characterizing free, unbound water
molecules [64, 65], which is reflected in the low-frequency absorption and re-
fraction spectra of water solutions [64,66,67]. It means that biological media

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consists of at least three components, namely biomolecules, free water and

bound to biomolecules water. Each fraction is characterised by distinctive
permittivity (εbm for biomolecules, εw for free water, and εbw for bound wa-

PT
ter). However, in the certain situations it is possible to neglect one of these
components, thus considerably simplifying calculations. For instance, in the
case of dry wood samples one can consider ε1 as the bound water permittivity

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and ε2 as wood cell walls permittivity, neglecting bulk water molecules [68].
However, in practice, multicomponent biological media are of strong in-
terest in practice. For instance, wood consists of air, water, and cell wall ma-

SC
terial (mixture of cellulose, hemicelluloses, and lignin) [68], while the retinal
nerve fiber layer involves axonal membranes, microtubules, neurofilaments,
and mitochondria [69]. Moreover, such components are structured in a cer-
tain hierarchy. Thereby one can set a task for creating a permittivity model

U
of different length scales.
AN
In many important cases, the biological medium consists of cells; where
their dimensions are much smaller than the THz wavelength. In addition, it
is possible that all of the cells in a tissue are prolate in a definite direction.
For instance, the length of tracheids reaches 3 mm for a pine and a spurce
M
and 10 mm for an agave. Whereas the tracheid cross-section linear size
is about 0.01 mm. Besides, one should take into account that each cell
is a complex multicomponent object. Meanwhile liquid water (practically
D

an inseparable part of a living cell) strongly absorbs THz radiation. For

example, a 1-mm-thick layer is essentially opaque over the entire THz range
TE

[70]. Therefore, it is not surprising that the most informative measurements

in the range 0.05–1.00 THz were made at a water layer thickness of 0.2 mm.
However, such a thickness is negligible with respect to tracheids length or
EP

the muscle tissue cell length. Therefore, comparatively thin sections are of
great interest for terahertz transmission spectral analysis. If one cuts the
sample perpendicularly to the tracheids (or to the similar prolate cells) such
a sample could be considered as non-periodical and homogeneous at least in
C

one direction. For the sake of simplicity, the chirality of the medium can be
neglected. In this case, one can consider the tissue permittivity model with
AC

a single preferential direction and two spatial scales [68].

The first spatial scale is related to cell walls with εcw and an intracellular
content with εic . The last one is considered as a homogeneous isotropic
media. Assuming that cells prolate in direction z, one can take into account
a dielectric anisotropy and use the following expressions for an anisotropic

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permittivity
εk = fcw εcw + fic εic , (26)
fic εic + (1 + fcw )εcw

PT
ε⊥ = εair , (27)
fic εcw + (1 + fcw )εic
where fic and fcw are the volume fractions of the cell walls and the intra-

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cellular content, respectively. The permittivity εk relates to the electric field
strength parallel to the long side of cells and the permittivity ε⊥ relates to
the electric field strength perpendicular to the mentioned direction.

SC
The second spatial scale is related to optical properties of the cell wall
and the intracellular medium defined by the properties of their components.
At this stage, one can use the effective medium approximation.

U
N1 N2
εcw,i − εcw εic,i − εic
= 0, = 0, (28)
X X
fcw,i fic,i
εcw,i + 2εcw εic,i + 2εic
i=1 AN i=1

where fcw,i = Vcw,i /Vcw , fic,i = Vic,i /Vic are the filling factor of i-th fraction,
Vcw,i and Vic,i are the volume of i-th fraction, Vcw and Vic are the volumes of
M
cell walls and intracellular content, respectively.
One can overcome the strict limitation of Bruggeman model, which is
valid for spherical form of constituents, by Polder and van Santen approach
D

for ellipsoidal particles [71]. Within this model the effective permittivity of
the two-component medium includes that of the host (εhost ) and elliptical
TE

inclusions (εparticle ) is
εhost
εeff = , (29)
1 3 1
1 − f (εparticle − εhost )
P
EP

3 i=1 εeff + (εparticle − εhost ) Ni

where f is the volume fraction of particles, Ni is i-th depolarization factor.

With x0 , y0 , z0 as an ellipsoidal axis lengths Nx takes form
C

x0 y0 z0 Z ∞ dξ
Nx = (30)
AC

.
2
q
0 (x20 + ξ) (x0 + ξ)(y0 + ξ)(z0 + ξ)

Analogue calculations deliver Ny and Nz . Developing Polder and van Santen

approach Scheller et al. deduced following formula for heterogeneous mixture

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permittivity [72]
−3N 2 + 3N
!

PT
!
εhost 3N + 1 εparticle − εeff
 
f =1− ×
εeff εparticle − εhost
12N − 18N 2
− 2
!

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(1 + 3N )εparticle + (5 − 3N )εhost
" #
9N 2 − 12N − 5
× , (31)
(1 + 3N )εparticle + (5 − 3N )εeff
where N = Nx , Ny = Nz = (1 − N )/2, f is the volume fraction of particles.

SC
At this point, the model describes only two-fraction medium of a host
material and embedded uniform particles. Moreover, these particles should
not be clustered. One can overcome these limitations taking into account

U
distributions of particle types and aspect ratios. Considering a mixture of
M different additives with a permittivity εpk , which are each divided into
AN
Lk groups of aspect ratios with a corresponding partial volume content fk,jk ,
the resulting total volume content of the additives in the mixture can be
described as  
M Lk
M
f= f˜k,jk  . (32)
X X

k=1 j=1

One can introduce the normalizing factor ψk,jk to take into account constant
D

relative volume content of each particle type

 
M Lk
TE

f˜k,jk = ψk,jk f, ψk,jk  = 1 (33)

X X

k=1 j=1

and value ζk,lk according to expression

EP

−3Nj2k + 3Njk
!
!
εhost 3Njk + 1 εparticle,k − εeff
 
ζk,lk= ×
εeff εparticle,k − εhost
C

12Njk − 18Nj2k − 2
!

(1 + 3Njk )εparticle,k + (5 − 3Njk )εhost

" #
9Nj2k − 12Njk − 5
AC

× . (34)
(1 + 3Njk )εparticle + (5 − 3Njk )εeff
Thus settling on the equations (31 - 34) one derives a valid description for a
mixture of particles with different ellipticity
 
M Lk
f =1− (35)
X X
 ψk,j k
ζk,jk 
k=1 j=1

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The developed approach is applicable as long as a quasi-static assumption is

justified [72].
The effectiveness of ”thin-layer” (TLA) and ”effective medium” (EMA)

PT
approximations in numerical simulation of dielectric spectra of biological cell
suspensions was verified in Ref. [73]. It was demonstrated that TLA and
EMA were both useful for greatly reducing computing while accurately fit-

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ting to analytical solutions. For a spherical cell or suspensions of spherical
cells aligned in a cubic array, dielectric spectra simulated by Finite-Element
Method (FEM) with TLA were in good agreement with those calculated from

SC
the Pauly-Schwan equation at volume fraction f < 0.25.
It is worth mentioning that analytical solution for permittivity is possible
in a case of simple object geometry. For example, in the case of dielectric
spectroscopy of single spherical cells [74]. In this paper, the permittivity

U
derivation is based on the Laplace transform of the single shell model for a
AN
spherical particle, and the full analytical expressions of the two characteristic
relaxation time constants have been derived. The suggested approach can
be extended to the multi-shell model.
Summarizing the above, effective medium theories are valid for describing
M
multicomponent media in a rather large amount of important cases, including
a description of biological tissues and fluids in biomedical applications of THz
science and technology.
D
TE

3 Time-domain and other terahertz spectro-

scopic techniques in biomedical diagnostics
EP

3.1 Terahertz time-domain spectroscopy

THz-TDS [75] is derived from time-domain spectroscopic techniques in
the microwave domain proposed in the 60s by Nicolson [76] and then by Nicol-
C

son and Ross [77]. The basic idea is to produce a repetitive train of ultrashort
electromagnetic pulses. The spectrum of each pulse spreads up typically to
AC

ν = 1/τ , where τ is the pulse duration. It follows that sub-picosecond pulses

exhibit a spectrum that extends over the THz range. Thanks to the avail-
ability of commercial femtosecond mode-locked lasers since the end of the
80s, such pulses are now commonly produced by rectifying the femtosecond
laser pulses.

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This is mostly achieved by photo-exciting free carriers in a semiconduc-

tor that exhibits a sub-picosecond free carrier lifetime, like low-temperature
grown GaAs [78], or by performing optical rectification in a nonlinear crys-

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tal, like ZnTe [79]. Both techniques are comparable in terms of performance.
With common non-amplified laser systems, photoconductive antennas are
maybe preferable, because they are more efficient in the low power regime.

RI
At higher laser powers, nonlinear crystals can be used to generate large THz
signals, when phase-matching between the laser and THz waves is obtained.
Thanks to the almost-perfect pulse train delivered by mode-locked lasers, a

SC
time-equivalent technique is used to record the THz waveform that impinges
the receiver. In the receiver, this THz waveform is mixed with a probe laser
pulse. In photoconductive antennas, the laser pulse generates free carriers
that are accelerated by the incoming THz field, and thus a photocurrent

U
proportional to the THz electric field is recorded through electrodes. In non-
AN
linear crystals, the THz pulse induces a birefringence that is read with the
probe laser pulse.
In any case, the recorded signal is equal to the convolution product of
the THz pulse and the optical effect in the antenna. By varying the relative
M
arrival times of the laser and THz pulses in the receiving antenna, with an
optical delay line, which is often a linear mechanical stage or an oscillat-
ing retroreflector on a voice coil, the temporal waveform of the THz pulse
D

is recorded. Thus THz-TDS setups look like a Mach-Zehnder interferome-

ter, in which a change from optical to THz frequencies occurs in one arm.
TE

Because emission and detection are triggered by the same laser pulse, the
equivalent time origin is well controlled and thus the THz signal is coher-
ent. Moreover, as the receiver collects signal only when excited by the laser,
EP

noise between two successive pulses is not recorded, and the experimental
dynamic range is very large. A numerical Fast Fourier Transform (FFT) of
the time domain pulse allows one to get the THz spectrum amplitude and
phase. Common THz-TDS setups, based on Ti-sapphire or fiber femtosec-
C

ond lasers, routinely deliver spectra ranging from 0.1 to 4.0–5.0 THz, with
a dynamic range exceeding 60 dB (even 90 dB when averaging) at the peak
AC

of the signal, i.e., around 0.5–1.0 THz. When using laser pulses shorter than
10 fs, frequencies above 10 THz and up to 200 THz [80] are generated, but
the spectra are not smooth and cannot be used to characterize samples over
10 THz [81, 82]. However, this THz-TDS characterization can be performed
up to 40 THz [83, 84] when generation and detection of the THz pulses is
achieved by air plasma breaking. The plasma is created by focusing an am-

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50 0

Spectral power density (dB)

40 -10
30 -20
Signal (nA)

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20 -30
10 -40
0 -50
-10 -60
-20

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-70
-30 -80
0 20 40 60 0 1 2 3 4 5 6 7 8
Time (ps) Frequency (THz)

SC
Figure 8: Typical THz pulse, and its spectrum, delivered by a THz-TDS
system with LT-GaAs antennas. (Courtesy of J.-L. Coutaz and IMEP-LAHC
laboratory, France)

U
plified laser pulse in air, with pulse energy of the order of mJ: the laser
AN
power density is so huge that it permits to both ionize air and accelerate
the charged particles, which radiate the THz signal [85]. Among different
schemes of the receiver, plasma-based detection is very efficient: the strong
M
THz pulse interacts with the photo-induced plasma to produce a second har-
monic optical beam, which is detected with a photomultiplier (namely the
ABCD technique, i.e., air based coherent detection [86]).
D

Some recent THz-TDS systems [87] employ two synchronized femtosec-

ond lasers: the repetition rate of one of them can be tuned, which replaces
the classical mechanical delay line and leads to very fast records. Different
TE

schemes have been proposed and are commercially available (Asynchronous

Optical Sampling (ASOPS), Electronically-Controlled Optical Sampling (ECOPS),
Optical Sampling by Cavity Tuning (OSCAT), etc. [67, 88–90]): usually the
EP

THz signal phase is controlled to a lesser degree than in very accurate classi-
cal settings, and therefore may be distorted, especially in THz-TDS reflection
measurements.
C

Spectroscopy of a sample is performed by putting the sample between

the emitting and receiving antennas, and measuring the detected waveform
AC

with and without the sample [91–93]. The ratio of the Fourier-transformed
waveforms gives the spectrum of the complex coefficient of transmission of
the sample. If the sample is flat with parallel faces and illuminated under
normal incidence, the complex coefficient of reflection t̃ (n, κ, ω) takes an
analytical expression that is numerically inverted to retrieve the complex

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refractive index ñ = n − jκ
ω
4ñ e−j c (ñ−1)d
t̃ (n, κ, ω) = (36)

PT
 ,
(ñ + 1)2 1 − ñ−1 e−j ωc ñd 2

ñ+1

where d is the sample thickness. The second denominator in Eq. (36) cor-

RI
responds to the Fabry-Perot effect – i.e., it describes back reflections of the
THz beam into the sample. The mathematical inversion of Eq. (36) is not
obvious because t̃ (n, κ, ω) exhibits an oscillatory behavior versus n or ω

SC
through the complex exponential term in relation Eq. (36). Method con-
sists of step-by-step variation of n and κ values for minimizing the differ-
ence t̃meas (ω) − t̃calc (n, κ, ω), but this procedure is a quite time-consuming
and needs heavy calculations, especially if it has to be repeated for each fre-

U
quency of the broadband THz spectrum. Therefore, smart inversion methods
AN
have been published. A first original inversion method has been proposed by
Duvillaret et al. [94] and is still widely employed. Basically, approximated
values na of n and κa of κ are easily obtained by omitting the Fabry-Perot
term in Eq. (36). Then, this Fabry-Perot term is approximately calculated
M
using na and κa . When divided by the Fabry-Perot term, Eq. (36) shows
much reduced oscillations and its solution is achieved by defining the error
function δ (n, κ) = (δρ)2 +(δϕ)2 , which zero is obtained by a steepest-descent
D

technique after a very few iterations. Here, δρ = ln (tcalc ) − ln (tmeas ) and

δϕ = ϕcalc − ϕmeas , where t and ϕ are respectively the modulus and phase
TE

of t̃. With modern personal computers, the determination of n and κ from

experimental data over the whole available THz spectrum is almost instan-
taneous. The method by Duvillaret has been refined by employing other
EP

error functions and zero-finding codes [95, 96]. A good review of extraction
methods is given in Ref. [97].
The precision of the determination of the studied sample parameters, i.e.,
n (ω) and κ (ω) (or the absorption coefficient α (ω) = 2 ωc κ (ω)), suffers from
C

two types of uncertainties, either due to random errors or to systematic errors.

Random errors, known as noise, is originated mostly by the laser intensity
AC

fluctuations (flicker noise, and laser and setup instabilities), the particle na-
ture of light (shot noise) both in emitting and receiving antennas, and the
electronic noise (Johnson noise, noise of amplification and analog-to-digital
conversion). The magnitudes of these different noise contributions, including
contribution of thermal fluctuations in the room, as well as rounding errors
in the Fourier transform, are shown in Fig. 9 that is adapted from Ref. [75].

29
 ACCEPTED MANUSCRIPT

The shot noise is by far the largest one, if the laser does not show specific
fluctuation noise due to a bad adjustment or worn components.

PT
RI
SC
Figure 9: Magnitudes (signal-to-noise ratio) for the main types of noises

U
that impact a THz-TDS signal. Here ADC stands for Analog-to-Digital
AN
Conversion, RIN for Relative Intensity Noise. (Courtesy of J.-L. Coutaz)

In a THz-TDS experiment, the THz pulse radiated by the emitting an-

tenna goes through the sample and then reaches the receiving antenna. The
M
noise in emission is thus transmitted through the sample and affects the
detected signal. Moreover, the noise in the detection antenna and in the de-
tection electronics adds to the emission noise. If σ is the standard deviation
D

of the signal, it is easy to show that

TE

σt2 = A (ω) t2 + B (ω) t + C (ω) , (37)

with A (ω) = (2σe2 + 2es ∆f + σd2 )/s2 , B (ω) = 2e∆f /s, and C (ω) = σd2 /s2 .
s = s (ω) is the amplitude of the THz signal delivered by the emitting
EP

antenna, σe is the noise of emission (mostly shot noise), σd is the signal-

independent noise of detection, and the ∆f terms corresponds to the Johnson
noise, where ∆f is the detection electronics bandwidth. Typically, for mod-
C

ern THz-TDS systems, A (ω) ∼ 10−5 , B (ω) ∼ 10−6 , and C (ω) ∼ 10−8 in the
vicinity of the maximum of the THz spectrum. Equation (37) permits calcu-
AC

lation of uncertainty ∆t of the transmission coefficient t for a given sample.

Then, by deriving expression t̃ = t ejϕ , we obtain ∆t = ∂∂ nt ∆n + ∂∂ κt ∆κ and
∆ϕ = ∂∂ nϕ ∆n+ ∂∂ ϕκ ∆κ, from which the noise-induced uncertainties ∆n and ∆κ
are derived (it is easy to show that ∆ϕ = ∆t/t). It follows that, as expected,
the errors ∆n and ∆κ depend on the transmission of the sample. Of course,
opaque samples stop most of the THz beam energy, and the extraction of

30
 ACCEPTED MANUSCRIPT

their refractive index ñ (ω) relying on a transmission-mode measurement ap-

pears to be rather imprecise. On the other hand, for moderately absorbing
samples, the uncertainty due to noise could be extremely weak, for example

PT
∆n < 0.1%. In this case, the actual uncertainty is much bigger as one has
to take into account systematic errors. Similar relations hold for THz-TDS
in reflection mode, where the transmission coefficient t̃ is substituted by the

RI
refectivity r̃. Finally, random errors may be minimized by integrating the
signal over longer times, if the TDS system and sample under study do not
exhibit long term drifts.

SC
Systematic (i.e., repeatable) errors include the effects of set-up misalign-
ment (sample illuminated by a non-parallel THz beam or at a slightly oblique
incidence, etc.), of the malfunctioning of system components (delay lines,
etc.), of a bad knowledge of the sample parameters (erroneous sample thick-

U
ness, sample faces that are not flat or not parallel, inhomogeneity, roughness,
AN
etc.). Also, errors could occur in the extraction technique, due to over sim-
plified models, numerical precision, etc. Contrary to noise, they cannot be
reduced by averaging the recorded signals. In Fig. 10, we show (left panel)
the contribution of the main sources of errors to the relative uncertainty
M
∆n/n for a 0.5-mm-thick sample featuring n = 2 and κ = 3 × 10−2 (i.e.,
α ≈ 13 cm−1 at 1.0 THz). The noise contribution is calculated for a com-
mon home-made setup. The sample is supposed to be tilted by 1◦ and its
D

thickness erroneous by 10 µm (2 %). Here, the main source of error of the

relative uncertainty ∆n/n is the noise, due to the rather large absorption in
TE

the sample. ∆κ/κ is of the same order of magnitude and depends mostly on
the thickness error. Typically, THz-TDS permits to determine n and κ with
a precision of few percents.
EP

When the sample is opaque, i.e., the transmitted THz signal is below
the noise threshold, it is compulsory to employ a reflection scheme. Here,
the requested reference signal is obtained by recording the signal reflected
by a metallic mirror. Reflection-mode THz-TDS is usually less precise than
C

transmission TDS, because of

AC

• a bad positioning of the reference mirror, even by a few microns;

• the signal of interest is given by the Fresnel coefficient at the sam-

ple surface, and is weaker than in transmission where it results from
propagation through the sample;

• an angular tilt of the sample and/or of the reference mirror, which

31
 ACCEPTED MANUSCRIPT

deviates slightly the reflected beam outside the receiving antenna [98].

In Fig. 10, we show (right panel) ∆n/n versus frequency for a sample with

PT
n = 2 and κ = 0.1 (α = 40 cm−1 at 1.0 THz). The sample is erroneously tilted
by 1◦ and shifted by 10 µm as compared to the reference mirror location.
These position errors, especially the deviation of the reflected THz beam,

RI
lead to enormous uncertainties. This deviation error may be corrected by
adjusting the sample orientation for achieving the maximal amplitude of the
reference waveform. In addition, several techniques have been proposed to

SC
minimize the shift error [99]. Typically, for highly absorbing samples, the
precision ∆n/n in reflection THz-TDS is 5–10 % and twice more for ∆κ/κ.
For transparent samples, errors are so large that the reflection THz-TDS
measurements are not relevant.

U
When dealing with liquids, one can use the rebound of the THz pulse
against the transparent window of the container as a reference pulse to miti-
AN
gate the positioning problem [100]. For soft materials and liquids, the atten-
uated total reflection (ATR) geometry is useful [95], because the face of the
prism is contact with the material makes its surface flat. Moreover, in ATR,
M
the magnitude and phase shift of the reflected signal are larger than that
in reflection-mode measurements. Other THz-TDS techniques include more
rarely polarimetry [101] and ellipsometry [102]. Pump-and-probe THz-TDS
D

permits to investigate the dynamics of free carrier population in semiconduc-

tors [103], of chemical reactions [104] [105], etc.
TE

1.E+02 1.E+04
1.E+03
1.E+01 mirror pos. angle: deviation
1.E+02
EP

angle: Fresnel noise

1.E+00 1.E+01
Dn/n (%)
Dn/n (%)

total
1.E+00
1.E-01 noise
thickness
1.E-01
angle
C

1.E-02 1.E-02

1.E-03 1.E-03
0 1 2 3 4 5
AC

0 1 2 3 4 5
Frequency (THz) Frequency (THz)

Figure 10: Typical error ∆n/n versus frequency in THz-TDS characteri-

zation: left panel shows moderately absorbing sample measured in trans-
mission; right panel shows highly-absorbing material measured in reflection.
(Courtesy of J.-L. Coutaz)

32
 ACCEPTED MANUSCRIPT

Samples can exhibit strong absorption bands due to some resonances

(vibro-rotation of molecules, phonons, etc.). When studied in transmission
mode, the THz signal could be below the noise level around the absorption

PT
peak, which means that the phase of the signal is not known in these spectral
bands. Therefore, for larger frequencies, the experimental phase could be
erroneously known by 2mπ, where m is an integer, and, thus, the refractive

RI
index is incorrectly estimated. This problem can be solved by

• performing simultaniously the reflection and transmission measurements

SC
[106];

• performing only the transmission measurements and treating the data

with a combined THz-TDS extraction procedure and a Kramers-Kronig

U
analysis [107];

AN
• using advanced methods of the THz-TDS phase correction [108]

In Fig. 11, we show (left panel) an example of combining the reflection-

mode and the transmission-mode THz-TDS measurements for analysis of
M
the maltose powder [106], and (right panel) an example of combining the

2.1 900 3.5 900

D

1.8 3
Refractive index n

Refractive index n
Absorption a (cm-1)

Absorption a (cm-1)
2.5
1.5 600 600
TE

2
1.2
1.5
0.9 300 300
1
0.6 0.5
EP

0.3 0 0 0
0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5
Frequency (THz) Frequency (THz)

Figure 11: Reconstruction of the refractive index n and the absorption co-
C

efficient α of samples featuring quasi-resonant lines of strong THz-wave ab-

sorption: left panel shows a THz response of the maltose powder, deter-
AC

mined by combining the reflection-mode and the transmission-mode THz-

TDS measurements; the dotted lines show the spectra obtained from only the
transmission-mode measurement; right panel shows a THz response of the
DAST crystal, determined by combining the transmission-mode THz-TDS
measurement and the Kramers-Kronig analysis. (Courtesy of J.-L. Coutaz)

33
 ACCEPTED MANUSCRIPT

transmission-mode THz-TDS measurement with the Kramers-Kronig analy-

sis for studying the DAST crystal (4-dimethylamino-N-methyl-4-stilbazolium
tosylate) [107]. From this figure, we can see that the absorption peaks of 100–

PT
500 cm−1 in magnitude are clearly determined.
Finally, many materials are not homogeneous or exhibit structural de-
fects, which induces scattering of the THz beam. Because the scattered light

RI
is kept within the sample or does not reach the receiver, absorption of the
sample can be overestimated. As the extraction procedure does not take it
into account, then scattering is interpreted as additional absorption. Apart

SC
from precisely measuring the scattered light in the whole half space behind
the sample, which is tricky and time-consuming if the scattering signal is
weak, the scattering contribution can only be evaluated from models and
from measuring different samples for which some parameters are varied, like

U
thickness, density of scatters, size of scatters, etc. The refractive index also
AN
might be affected by the scattering phenomenon. When the size of the scat-
tering particles is much smaller than the involved THz wavelengths, the scat-
tering can be neglected. The material can be considered as a homogeneous
material, and its dielectric response is well described by the abovementioned
M
effective medium theories (like the Maxwell-Garnett [109], the Polder and
Van Santen [72], the Bruggeman theories [110]) or by the power law ap-
proaches (like the Landau-Lifshitz-Looyenda model [111] or the Lichtenecker
D

and Rother model [112]).

When scattering occurs, three different regimes can be defined.
TE

• The Rayleigh scattering (λ < 10d, where d is a particle diameter), for

which the effect of the particle shape is not significant, features the
scatterinf efficiency of ∝ λ−4 .
EP

• The Mie scattering on a medium-size particles (0.1d < λ < 10d), is

characterized by the scattering efficiency of about ∝ λ−2 for s single
scattering of photons in a sample; this scattering regime strongly de-
C

pends on the particle geometry.

AC

• The scattering in a medium comprised of large-scale particles (λ > 10d)

allows for describing the response of a medium using the principles of
geometric optics. For this scattering regime, some simple phenomeno-
logical models have been proposed to avoid computing the Mie scatter-
ing cross-sections. As an example, the Raman model of the so-called
Christiensen scattering effect was introduced; it involves a statistical

34
 ACCEPTED MANUSCRIPT

100

Contribution of scattering (%)

80

PT
60

40

RI
20

0
0 100 200 300 400 500 600

SC
Particle size (µm)

Figure 12: Contribution of scattering to the extinction of the THz

beam, propagating through a powder mixture of fructose and High-Density

U
Polyethylene (HDPE), versus the fructose grain size. (Adapted and repro-
duced from the Ref. [113], with the permission of AIP Publishing)
AN
calculation of optical re-reflections in 1D multi-layered structures, and
leads to:
M
2π
 2
α = αabs + αscat = αabs + K (npart − nmatrix ) d, (38)
λ
where K is a numerical parameter, depending on the shape, concen-
D

tration and distribution of particles. Equation 38 works well for small

concentrations of scatterers.
TE

In Fig. 12, we show a contribution of scattering to the extinction of the

THz beam, passing through a powder mixture of fructose and High-Density
Polyethylene (HDPE) [113]. Here, the frequency is 1.5 THz and the exper-
EP

imental data are plotted versus the fructose grain size. The continuous line
is calculated using a Mie-scattering code. In the Rayleigh scattering regime
(i.e., for the grain sizes below 20–30 µm), the scattering contributes to less
than 10 % of losses; thus, it can be neglected. In the Mie scattering regime
C

(i.e., for the grain size of 100 to 500 µm), the scattering is resonant and
causes more than 70 % of the THz beam extinction.
AC

3.2 Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy was the main tool to
perform spectral studies in the far-infrared band up to the invention of THz-
TDS, and it is still widely used in laboratories because commercial systems

35
 ACCEPTED MANUSCRIPT

Fixed mirror

Moveable mirror

PT
Source
Beam x
splitter

RI
Detector

SC
Figure 13: A scheme of the FTIR spectrometer. (Courtesy of J.-L. Coutaz)

U
are robust, easy-to-use and exhibit good performance. Most FTIR spectrom-
eters are based on a broadband radiation sources and a Michelson interfer-
AN
ometer, in which the optical length of one arm is varied by moving its mirror
(see Fig. 13).
It is easy to show that the signal delivered by the detector is
M
+∞
ω
Z   
S (x) = Io (ω) T (ω) R (ω) 1 + cos 2 x dω, (39)
c
0
D

where Io (ω) is the spectral flux of the source; ω is the optical frequency; T (ω)
is the transmission of the interferometer taking into account the spectral
TE

response of the mirrors, beam splitter, etc.; R (ω) is the spectral responsivity
of the receiver; x stands for the position of the mirror. The signal S (x)
defined by the Eq. (39) is called interferogram or waveform, because the
EP

distance x is proportional to the propagation time (x = ct). It is obvious that

the interferogram is proportional to the Fourier transform of the spectral flux
of the source; thus, this spectral flux Io (ω) is obtained by taking the inverse
Fourier transform of S (x). As compared to dispersive spectroscopic schemes,
C

such as grating spectrometers, FTIR spectrometry shows two advantages.

AC

• The Fellgett advantage: All the spectral components, for a given value
of x, are recorded simultaneously leading√to a large signal. If the setup
is limited by the shot noise, the SNR is M better than with disper-
sive spectrometers, where M is the number of the recorded spectral
components.

36
 ACCEPTED MANUSCRIPT

• The Jacquinot advantage: In dispersive spectrometers, the spectral

resolution is inversely proportional to the width of the entrance and
output slits, while in FTIR spectrometers, it is inversely proportional

PT
to the maximum displacement of the moveable mirror. Therefore, the
throughput of FTIR spectrometers is better than that of the dispersive
ones.

RI
To adapt the FTIR method for measurements in the THz band, it should
include source, detector and beams splitter, capable for operation at THz
frequencies. Generally, the broadband source is a mercury lamp, in which

SC
both the mercury vapor and the bulb quartz envelop radiate a blackbody-like
spectrum, and the cryogenic detector is either a InSb hot-electron bolometer,
or a superconducting bolometer, or a germanium photoconductor. However,

U
most commercial FTIR spectrometers address the mid-infrared range, and
only few of them operates below 1.0 THz.
AN
Also, the performance of the FTIR spectrometers is determined by the
quality of the beam splitter: it should preferably be 50/50 % and transparent
over a broadband spectral range, and work at a 45◦ oblique incidence without
inducing special spectral features. Very thin (≤ 10 µm) boPET (biaxially ori-
M
ented polyethylene terephthalate, commercialized as Mylar) films that could
be multi-layered or coated with a nanometric-thin metal layer, are commonly
employed. Another elegant solution is to employ the Martin-Puplett scheme,
D

in which a wire grid polarizer serves as a beam splitter. However, the polar-
ization dependance of the wire grid reflection/transmission obliges to rotate
TE

the THz beam polarization in one arm of the interferometer. This is realized,
at the expense of a more complex layout and of a detected power divided
by two, by substituting the common flat mirrors in each of the two arms by
EP

top-roof mirrors aligned perpendicularly.

FTIR devices are mostly employed as a kind of monochromator: the
sample is located at the output of the interferometer, in front of the detector.
C

Only its transmittance T (ω) is measured versus frequency. When the sample
is rather absorbing, the contribution of Fresnel losses at its surface may be
AC

neglected, and the absorption coefficient of the material is deduced from the
Beer-Lambert law as α (ω) = − ln T (ω) /d. The example of water vapor
(pressure 20 Torr) measurements is given in Fig. 14.
If the sample of thickness d is transparent, its transmittance varies as
8n2
T (ω) =  . (40)
n4 + 6n2 + 1 − (n2 − 1)2 cos 2 ωc nd

37
 ACCEPTED MANUSCRIPT

2.5
1.0
2.0
0.8
1.5

Transmittance
Signal (A. U.)

PT
0.6
1.0
0.4
0.5

RI
0.0 0.2

-0.5 0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.5 0.7 0.9 1.1 1.3 1.5
Distance x (cm) Frequency (THz)

SC
Figure 14: FTIR spectrum of water vapor (20 Torr, 1-m long path): left
panel shows interferograms without (bottom curve in blue) and with (top
curve in red, shifted in a vertical direction) water vapor; right panel shows a

U
transmission spectrum in 0.5–1.5-THz-range. (Courtesy of J.-L. Coutaz)
AN
This transmission exhibits a periodic behavior with period ∆f , from which
the refractive index could be deduced as n = c/ (2d∆f ), in case if n does not
vary strongly within the interval ∆f . A smarter way to characterize a mate-
M
rial (i.e., to reconstruct its refractive index n and absorption coefficient like
when using THz-TDS [114]), is to put the sample in one arm of the interfer-
ometer. This technique, named Dispersive Fourier Transform Spectroscopy
D

(DFTS), delivers both magnitude and phase of the sample transmittance as

in THz-TDS. Therefore, extraction codes similar to those used in THz-TDS
TE

are used to determine the sample optical properties. However, DFTS is not
so popular because the adjustment of the setup is very delicate.
Finally, how could we compare FTIR and THz-TDS? With modern FTIR
EP

and THz-TDS components, the recording times are similar. In THz-TDS

systems, the signal is not recorded between pulses; thus, noise is not time-
integrated. This makes FTIR noisier than THz-TDS. For example, Han et
al. [79] reported that below 3.0 THz, FTIR shows about five-orders weaker
C

SNR than that of THz-TDS. In addition, FTIRs usually operate rarely below
1.0 THz; but on the other hand, they sweep much higher frequencies than
AC

THz-TDS. Extraction of both refractive index and absorption coefficient is

easy based on the THz-TDS data; while it requires the use of the Kramers-
Kronig transform when working with the FTIR data; the latter could lead
to undesired edge effects (or Gibbs noises). Thus, FTIR method is more
adapted to determine the transmission spectra and absorption coefficients of

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200
THz-TDS
160 FTIR

PT
Signal (A. U.)
120

80

RI
40

0
0 1 2 3 4 5

SC
Frequency (THz)

Figure 15: An absorption coefficient of a mixture of 75 % maltose and 25 %

HDPE powders, measured by THz-TDS and FTIR. (Adapted from the Ref.

U
[115])

AN
samples. In Fig. 15, we show an absorption coefficient of a mixture of 75 %
maltose and 25 % HDPE powders, measured with a home-made THz-TDS
and a commercial FTIR spectrometer (Brucker Vertex) [115]. The curves are
M
very similar. Over 3.0 THz, the THz-TDS signal vanishes, while FTIR still
delivers a useful spectrum. On the other hand, at resonance, FTIR leads to
an under-estimated absorption due to a contribution of the neglected Fresnel
D

lossess.
TE

3.3 Raman spectroscopy

The Raman effect (also known in Russia as the Landsberg-Mandelstam
effect or combinational scattering) and Brillouin effect correspond to the
EP

transfer of a small amount of energy of the incident photons to matter via

inelastic scattering. While in Brillouin scattering, the transferred energy cor-
responds to microwave frequencies, and is due to the incident light scattering
C

of phonons, Raman scattering involves energies spreading from the THz up

to the mid-infrared as the incident light interacts with vibrational modes.
AC

Therefore, Raman scattering is another way of studying the far-infrared re-

sponse of matter. This transfer of energy to the molecules of the illuminated
matter corresponds an inelastic scattering described by the following energy
conservation equations:

~ωStokes = ~ωlaser − ~ωres , ~ωanti−Stokes = ~ωlaser + ~ωres , (41)

39
 ACCEPTED MANUSCRIPT

where ~ωres is the difference between two energy states of the molecule, while
scattered Stokes and anti-Stokes signals differ by a higher or lower photon
energy, i.e., adding or subtracting ~ωres to or from ~ωlaser . Thus, in the

PT
Stokes phenomenon, the molecule is excited by the laser beam to a virtual
energy state, and then it returns not to the fundamental state, but to a vibra-
tional/rotational energy level. In the anti-Stokes phenomenon, the molecule

RI
is initially in a vibrational/rotational energy level and after excitation it re-
turns to the fundamental state. Therefore, a Raman spectrum shows spectral
lines symmetrically apart from the laser frequency; the Stokes and anti-Stokes

SC
intensities are governed by the population of the vibrational/rotational en-
ergy level – i.e., by a Maxwell-Boltzmann distribution
Ianti−Stokes

U
= e−~ωres /kB T . (42)
IStokes
AN
It follows that for resonances occurring in the THz range and at room tem-
perature, the intensities of the Stokes and anti-Stokes signals are of the same
order of magnitude. The resonances involved in Raman scattering are molec-
ular vibrations, rotations or any more complex deformation of the molecule
M
skeleton.
The Raman effect, and its difference/similarity with THz spectroscopy,
can explained by a very simple classical model, whose results are in agreement
D

with what is obtained from quantum mechanics calculations. Let us consider

a molecule that possesses a dipole momentum p~ induced by an exciting light
TE

beam:
p~ (t) = α : E
~ (t) = α :E
~ light ejωlight t , (43)
↔ ↔

where α is the polarizability of the molecule, that must be a tensor for the
↔
EP

molecule to be Raman active; α depends on the position Q ~ i of each atom i

↔

of the molecule. At non-null temperature, these positions vary slightly with

time due to the thermal agitation, and, thus, the polarizability is function
C

of time α (t). Nevertheless, the thermal movement is very weak, and, thus,
↔

α (t) can be expanded in a Taylor series

↔
AC

↔
∂α
α (t) = αo + dQi (t). (44)
↔ ↔
X

i ∂ Qi

When the movements of the atoms correspond to a resonance, each atom

i is oscillating around its equilibrium position at a frequency ωres , such as

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 ACCEPTED MANUSCRIPT

dQi (t) = Qoi ejωres t . Entering the expression of α (t) in Eq. (43) leads to
↔

↔
!
∂α ~
p~ (t) = αo : E
~ light e + : Elight ej (ωlight ±ωres )t . (45)
↔ jωlaser t
X

PT
Qoi
i ∂ Qi

This expression of α (t) explains the Raman effect and illustrates its differ-
↔

RI
ence with THz spectroscopy. The first right-side term of Eq. (45) leads to the
susceptibility of matter that is addressed in THz spectroscopy (ωlight = ωTHz ).
The second term of Eq. (45) describes the Raman effect, both Stokes and anti-

SC
Stokes phenomena depending on the sign of ±, which involves the resonance
of the molecule at a frequency ωres = ωTHz . Therefore, THz spectroscopy will
study the polarizability of molecules at THz frequencies, while Raman effect
is related to the derivative of this polarizability versus the local coordinates

U
of the molecule. It follows that both techniques give complementary informa-
tion about the electromagnetic response of matter. If αo shows some specific
↔

AN
spectral features due to resonances, its derivative will also be exhibits some
peculiarities at the same frequencies, but the THz and Raman spectra could
also be very different. In addition, when performing a rigorous quantum
M
mechanics analysis, it appears that both phenomena obey different selection
rules: typically, when considering molecular vibrations, a molecule is active
if its dipole moment varies during the vibration, while it is Raman-active if
D

its polarizability change. This explains why the ν1 vibration mode of CO2 is
Raman-active, but does not show any specific absorption feature. Selection
TE

rules depend on the involved resonances: for example, transitions between

vibrational levels of a diatomic molecule are governed by ∆ν = ±1, while
rotational levels of of linear molecules obey ∆J = ±2.
The main advantages and disadvantages of Raman spectroscopy, as com-
EP

pared to THz spectroscopy, are the following.

Advantages
C

• It does not require pulsed or tuneable laser. A laser delivering a short

wavelength is preferable because the Raman efficiency varies as λ−4 .
AC

• The rotational and vibrational spectra of several different species can

be recorded simultaneously.

• The Raman signal intensity varies linearly with the pumping laser in-
tensity, and no saturation effect is observed.

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• The signal intensity is proportional to the Raman cross-section, which

is larger for molecules with extended π-electron systems, with large,
electron-rich atoms, such as S or I, or with multiple bond stretches.

PT
Thus Raman spectroscopy is well suited for major molecules like N2 ,
O2 , CO, CO2 , H2 O, etc., but not for molecules with single C-H, C-O
and C-C bonds.

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• The Stokes and anti-Stokes wavelengths belong to the UV-VIS domain
when the pumping laser is also UV-VIS. Thus the map of a Raman

SC
signal radiates by a sample could be achieved by scanning the laser
beam with a sub-micronic spatial resolution.
Disadvantages

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• The Raman signals are weak (about 1000 times weaker than Rayleigh
scattering), therefore Raman spectroscopy requires high power lasers,
AN
which can heat and degrade the sample, especially in the CW regime.
• Raman spectroscopy is sensitive to background fluorescence and emis-
sion, and stray light.
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Also, laser pumping in Raman spectroscopy is preferably performed at a short
wavelengths (NIR, VIS or UV), which is very different from the THz range
D

and constitutes either an advantage or a disadvantage. For example, liquid

water can be easily characterized by Raman spectroscopy, because water is
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almost transparent at the laser and Stokes frequencies. THz spectroscopy

of liquid water is more difficult to perform because water absorbs the THz
waves.
EP

In Fig. 16, we show the THz absorption coefficient and the the Raman
signal intensity for the cysteine (left panel; adapted from [116]) and the crys-
talline indapamide (right panel; adapted from [117]) The spectra for cysteine
exhibit similar features, for example, the peak at 1.65 THz (556 cm−1 ) that
C

are attributed to S-S bridges; while the spectra for the crystalline indapamide
(i.e., the drug for the treatment of hypertension) notably differ.
AC

Thereby, we can conclude that the spectroscopic study of the far-infrared

response of water and hydrated samples is efficiently performed with THz-
TDS, which allows one to determine the complex refractive index of the
samples, and permits also polarimetric, ATR, and time-resolved pump-and-
probe experiments. Raman measurements are complementary to THz-TDS,
providing an additional information on the studied molecules. FTIR method

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200 200
THz-TDS THz-TDS
160 Raman 160 Raman

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Signal (A. U.)
Signal (A. U.)

120 120

80 80

40 40

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0 0
0 1 2 3 4 5 6 7 0 1 2 3
Frequency (THz) Frequency (THz)

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Figure 16: The THz absorption coefficient and the Raman signal intensity
for the cysteine (left panel; adapted from the Ref. [116]) and the crystalline
indapamide (right panel; adapted from the Ref. [117]).

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delivers only the absorption spectra of the samples, which could be sufficient
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for restricted studies.
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4 Modern terahertz instrumentation for ap-
plications in biology and medicine
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4.1 Introduction
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In the THz range, efficient sources and detectors still remain rare, expen-
sive and cumbersome. The development of the THz components is difficult
because this range is located in the gap between the realms of optics and elec-
tronics, where the related electronic and optical principles of electromagnetic-
EP

wave generation and detection do not work properly. On the electronic side,
the efficiency of components is limited by RC time-constant effects; while, on
the optical side, it is limited by thermal effects. In this section, we start with
C

a brief review of modern tendencies in the area of highly-efficient THz-wave

generation and detection, with an emphasis on the THz devices, capable for
AC

use in the THz biophotonics.

Another problem, significantly restricting the list of biomedical applica-
tions of THz technology, is a low diffraction-limited spatial resolution of the
majority of modern lens- and mirror-based THz optical systems. To mitigate
this problem, numerous novel modalities of THz spectroscopy and imaging
with the spatial resolution beyond the λ/2-Abbe-limit are vigorously ex-

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plored nowadays. We review modern methods of sub-wavelength-resolution

THz imaging and discuss their prospectives in biology and medicine.
Finally, we consider another circumstance, limiting reliability of THz

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technology, which is associated with the lack of efficient THz optical waveg-
uides and endoscopes, capable for THz-wave delivery to hard to access bi-
ological objects, tissues and internal organs. We describe modern trends in

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the THz waveguiding technology and analyze applicability of the advanced
THz waveguides in biophotonics.

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4.2 Advances in opto-electronics sources and detectors
coming to the terahertz range

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A wide variety of physical processes can emit THz waves [118–123], for
example, vacuum electronic systems, including klystron, traveling-wave tube,
AN
gyrotrons, free electron lasers, synchrotrons and backward-wave oscillators
[118, 124, 125]. Laser type sources include gas lasers, semiconductors and
quantum cascade lasers (QCLs). As an example, in Fig. 17, we show a
schematic of NH3 gas laser, which is pumped by a mid-IR QCL instead of a
M
conventional CO2 laser pump [126]; this profits from the tunability of QCL;
thus, giving access to new laser lines, which are not accessible when using a
CO2 laser pump. In the discussed work, the authors demonstrated a laser
D

emission at 1.07 THz with the average power of 34 µW, the low-threshold of
2 mW, and the impressive differential efficiency of 0.82 mW/W.
TE
C EP
AC

Figure 17: NH3 gaz laser pumped with a mid-IR beam. (Reprinted from the
Ref. [124])

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Another category of sources is further defined by the fact that they are
pumped by optical lasers. Some are continuous, like photomixing, and others
pulsed. The most used technique is photoconductive antennas, but many oth-

PT
ers techniques like bulk or surface optical rectification, air plasma generation
magnetic dipoles and parametric oscillators can also be used [127]. Recently,
significant increase in the optical-to-THz-wave conversion efficiency was re-

RI
ported for the THz photoconductive antennas, the performance of which was
boosted using plasmonic and/or dielectric nanoantennas incorporated into a
photoconductive gap [128]. In such photoconductive antannas, a strong con-

SC
finement of the optical pump behind the nanoantennas leads to enhancement
of the light – matter interaction, reduction in the lifetime of photoexcited car-
riers, and improvement of the thermal efficiency of the antenna [128–130].
Using different geometries of metal and/or dielectric nanoantennas, such as

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silver nanoislands or arrays of nanoscale apertures [131, 132], monopole or
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dipole plasmonic gratings with either low or high aspect ratio [133–140], plas-
monic nanocavities combined with Bragg reflectors [141], metal colloidal par-
ticles deposited onto the photoconductive substrate [142], and all-dielectric
gratings [143], an impressive enhancement (by orders of magnitude) of the
M
output power of photoconductive emitters and the sensitivity of photocon-
ductive detectors was demonstrated.
At lower frequencies, sources from the electronic world have the advantage
D

of being very compact. For example, GaAs Schottky diodes are developed for
many years with a continuous increase of the power and the sensitivity; they
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have many applications as frequency multiplicators and detectors [145]. In its

C EP
AC

Figure 18: A current response of the integrated SiGe Heterojunction Bipo-

lar Transistor (HBT) near-field sensor for differently concentrated lactose
solutions. (Reprinted from the Ref. [144])

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turn, GaN high-electron-mobility transistors is another example of electronic

system that can be used for THz-wave generation or detection [146]. In the
past decades, there has been an increase of the power and the frequency of

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electronic systems, as well as the standard silicon CMOS techniques, which
are no longer confined to millimeter waves, but extend to the THz domain.
The Complementary Metal-Oxide-Semiconductor (CMOS) technology allows

RI
for creating the low-cost arrays of THz sources and detectors [147]. Further-
more, the CMOS technology permits new integrations and designs such as
near-field sensors compatible with liquid and biomedical environments. In

SC
Ref. [144], a THz near-field sensor was developed to measure biologically rel-
evant components in aqueous solutions; This sensor uses 0.13 µm SiGe HBT
technology and operates at 0.53 THz. From Fig. 18 we see that this sensor
yields characterization of concentration of lactose dissolved in de-ionized wa-

U
ter. The main advantage of this near-field sensor is that it combines low-cost
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and compact implementation with high-sensitivity. Finally, it can be fabri-
cated in a form of an array with a ∼ 1 µm spatial resolution suitable for
high-resolution THz bioimaging.
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4.3 Overcoming the diffraction limit and increasing
the spatial resolution
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Biomedical applications of THz technologies suffer from low spatial reso-

lution of THz spectroscopy and imaging, which is primarily limited by diffrac-
TE

tion limit being the function of electromagnetic wavelength λ and numerical

aperture NA of the optical system [148]:
λ
EP

δ= , NA = n sin σ 0 , (46)
2NA
where n is the refractive index of medium, in which the beam caustic is
formed, and σ 0 is an aperture angle. The structural components of tissues
C

(microfibrils, cells, cell organelles, etc.) are features that have much smaller
dimensions compared to the THz wavelength λ; thus, they could not be vi-
AC

sualized using conventional THz instruments. Overcoming the diffraction

limit and bringing the resolution of THz imaging and spectroscopy to sub-
wavelength scales remain an important problem of THz science and technol-
ogy.
In this section, we discuss the existing techniques of THz spectroscopy
and imaging from the viewpoint of their spatial resolution and applicability to

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THz measurements in tissues. For this purpose, in Fig. 19, we schematically

plot the spatial scales of tissue components d [149] versus the spatial resolu-
tion of modern THz spectroscopy and imaging systems δ, where d and δ are

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normalized by the characteristic electromagnetic wavelength λ0 = 300.0 µm
corresponding to the electromagnetic frequency ν0 = 1.0 THz.
The majority of modern THz spectroscopic and imaging system rely on

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the use of single-element lenses and off-axial parabolic mirrors [150], the
spatial resolution of which is limited not only by the diffraction limit, but

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D
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AC

Figure 19: A comparison of scales in tissues d (see the top part of the fig-
ure) with the spatial resolution of modern THz spectroscopy and imaging
techniques δ (see the bottom part of the Fig.). For simplicity, d and δ are
normalized by the characteristic electromagnetic wavelength λ0 = 300.0 µm
corresponding to the electromagnetic frequency ν0 ' 1.0 THz. (Courtesy of
K.I. Zaytsev)

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also by the wavefront aberrations and the dimensions of the optical sys-
tem [151]. Conventional spherical lenses and off-axial parabolic mirrors fea-
ture typical numerical aperture of NA = 0.4...0.5 and yield spatial resolution

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of δ ' 1.2...1.0λ. As shown in Fig. 19, the scales of tissue components are
much smaller compared to this spatial resolution; thereby, the single spher-
ical lenses and off-axis parabolic mirrors allow studying only the effective

RI
response of tissues averaged within the beam spot (≥ λ2 ). Further improve-
ment of the single spherical lens performance by increasing its aperture and
resolution might be challenging owing to significant residual aberrations since

SC
the spherical surfaces do not have much potential for correcting the wave-
front aberrations at higher apertures [152]. The off-axis parabolic mirrors
suffer from overlapping of incident and focused beams when operating at
high numerical apertures, which also limits the size of the beam spot [150].

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Aspherical singlets allow improving the numerical aperture and the spatial
AN
resolution of THz measurements to about NA = 0.6...0.7 and δ = 0.8...0.7λ,
respectively, thanks to high potential of aspherical optical surfaces for cor-
recting the wavefront aberrations [150, 152]. When operating at such a high
numerical apertures, the aspherical singlets feature small field of view [152],
M
which limits their applications in THz imaging only with point-by-point scan-
ning of the sample. The aspherical optical elements almost reach the Abbe
diffraction limit [151] of the electromagnetic beam focusing, which can be de-
D

fined for the largest-possible numerical aperture in free space NA = sin 90◦ =
1.0 as
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λ
δ≥ . (47)
2
In Fig. 19, we show that the THz instruments relying on the aspherical optics
only measure the spatially-averaged effective properties of tissues on the cel-
EP

lular level [149]. Neither spherical and aspherical lenses, nor off-axis parabolic
mirrors allow us to overcome the Abbe limit and, as a consequence, visual-
izing sub-wavelength features of tissues is not possible using conventional
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optics.
Several methods of THz imaging with the resolution beyond the Abbe
AC

limit using lens-based optical systems have been developed exploiting the
effects of electromagnetic field confinement at the shadow side of various
obstacles/particles placed in front of the focal plane [153]. Among them,
the THz solid immersion imaging uses so-called solid immersion lenses (SIL)
[154]. As shown in Fig. 20 (a), the SIL is typically formed by placing a
truncated sphere, which is made of material possessing high refractive index

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RI
SC
Figure 20: THz SIL imaging: (a) a scheme of the THz SIL comprised of
the HDPE wide aperture aspherical singlet and the HRFZ-Si hemispherical

U
lens [154]; (b) a comparison of the normalized THz beam spots formed by
the THz SIL and the separately-operating aspherical singlet. Panel (b) jus-
AN
tifies δ = 0.35λ0 -resolution of the THz SIL imaging, where λ0 = 600.0 µm
is the electromagnetic wavelength of CW THz radiation employed in the
experiment. (Courtesy of K.I. Zaytsev)
M
nSIL , between a wide-aperture focusing objective and the image plane. An
electromagnetic wavelength is nSIL times smaller in the truncated sphere, as
D

well as in an evanescent field volume behind it, than those in the free space.
By forming the THz beam caustic in the free space, at a small distance from
TE

the truncated sphere, we are able to achieve up to nSIL -times reduction in

the dimensions of the beam spot compared to that observed in case of an
ordinary focusing in a free space. When using the high-resistivity float-zone
silicon (HRFZ-Si) as a truncated sphere material, we are able to achieve
EP

nHRFZ−Si ' 3.42 times increase of the spatial resolution.

In Fig. 20 (b), we show experimental data for the normalized THz beam
spot formed by THz SIL, operating in CW mode and comprised of an aspheri-
C

cal singlet, made of HDPE, and a hemispherical lens, made of high-resistivity

silicon manufactured using float zone method (HRFZ-Si) [154]; here, the THz
AC

beam spot formed by the separately-operating aspherical singlet is shown as

a reference. The observed results justify δ = 0.35λ-resolution of the THz
SIL, which is much higher compared to δ = 0.85λ-resolution provided by the
separately-operating aspherical singlet. In some recent work [155–157], an
approach for handling of soft tissues at the focal plane of THz SIL was pro-
posed, and the spatial resolution of THz SIL was further improved to 0.15λ,

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which represents a considerable advancement over all configurations of SILs,

reported previously in millimeter-wave, THz, IR and visual bands. The THz
SIL imaging has a potential for visualization large-scale components of tis-

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sues, such as microfibrils and large cells (see Fig. 19); thus, promising novel
applications in biology and medicine.
Another approach allowing one to slightly overcome the Abbe limit is

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based on generation of so-called Tera-jets – i.e., local sub-wavelength scale
caustics of the THz field formed by placing a mesoscale dielectric particle
(cubic, spherical, pyramidal, or others) at the focused imaging point [158].

SC
The spatial resolution of Tera-jet imaging can reach δ = 0.4...0.5λ, which
makes possible visualization of fibrous tissues and large cells (see Fig. 19). At
the same time, the Tera-jet imaging is characterized by the limited spectral
range, which reduces its reliability for THz biomedicine, since the majority of

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THz biomedical applications require the use of broadband THz wave sources.
AN
The THz holography [159] and synthetic aperture imaging [160] demon-
strate another approach for bringing the resolution of THz imaging resolution
to slightly sub-wavelength scale. The wide aperture holography seems to be
rather competitive, and it has been already applied for imaging of tissues;
M
for example, see Ref. [161]. At the same time, it requires complicated com-
putational algorithms for resolving the ill-posed inverse problems [162, 163].
Despite the resolution enhancement provided by the described THz SIL,
D

Tera-jet, holography and synthetic aperture imaging modalities, they are un-
able to provide essentially sub-wavelength spatial resolution and, as a con-
TE

sequence, an ability for studying micro- and nano-scale objects. This pushes
further developments into the realm of the lens-less near-field THz imaging.
The THz near-field scanning-probe microscopy allows one to overcome the
EP

Abbe diffraction limit, providing spatial resolution of δ = 0.1λ [164] or even

δ = 0.001λ [165–167]. In Fig. 21, we show a representative scheme of the
THz near-field scanning-probe microscope, which is based on the use of the
metal tip (cantilever) as a near-field probe providing deep-sub-wavelength
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confinement of the incident THz field. The THz field observed after scatter-
ing by a small probe contains information about the physical properties of
AC

microscale (and even nanoscale) features of the object, near which the probe
is located.
The THz near-field microscopy has been recently applied to the THz
imaging of tissues (for example, see Ref. [168]); it has potential for visualiza-
tion microfibrils and blood vessels, separate cells and even cell organelles (see
Fig. 19). While capable of superior resolution, the THz near-field imaging

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RI
SC
Figure 21: A scheme of the THz near-field scanning-probe microscope, which
employs the metal tip (cantilever) as a near-field probe providing local en-
hancement of the THz field scattered on a microscale (or a nanoscale) frag-
ments of the object. This scheme is similar to the one from the Ref. [166],

U
where the modulation of the vertical position of the probe h (t) = h (t + T )
(T is a period of the modulation) and the demodulation of the detected THz
AN
scattered field Esca (t) = Esca (t + T ) was used in order to increase the signal-
to-noise ratio and achieve the depth information about the object. (Courtesy
of K.I. Zaytsev)
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is plagued with several disadvantages. Firstly, it requires a small working

distance thereby, the scanning probe (tip or diaphragm), which is placed in
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the object plane, may interact with the sample and perturb its structure.
Secondly, detection of light confined at, and scattered by, very small tips, or
TE

transmitted through sub-wavelength diaphragms, requires rather powerful

emitters and highly sensitive detectors, which are still rare and expensive.
Third, registration of a single THz near-field image seems to be rather time-
EP

consuming procedure. These disadvantages make the THz scanning-probe

near-field imaging predominantly a tool of laboratory research.
In Ref. [169], an approach for reduction of the THz wave energy losses
in the THz near-field microscopy has been proposed. In order to enhance
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the THz wave transmission through the sub-wavelength diaphragm, it relies

AC

on the use of the bowtie shaped aperture surrounded by concentric periodic

structure in a metal film – i.e., a bull’s eye structure. The THz near-field
imaging system based on such an aperture was reported to provide λ/17 spa-
tial resolution. In particular, it was used for visualization of the 20-µm-width
sub-wavelength metal pattern at λ = 207 µm. Another improvement of the
THz near-field imaging is associated with the use of the optical superlenses

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based on negative index or plasmonic materials. For example, in Ref. [170],

the authors construct a superlens based on graphene monolayer with a grat-
ing voltage gate. This lens operates based on the Fabry-Perot resonance of

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plasmon waves. It yields obtaining magnified images of sub-wavelength pat-
terns in the frequency range of 4.3 to 9.0 THz with the resolution reaching
λ/150.

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The THz imaging based on wire media (WM) [171, 172] represents an-
other remarkable techniques of THz near-field imaging, which has a poten-
tial to provide spatial resolution up to δ = 0.1...0.5λ [171] (see Fig. 19).

SC
For THz image formation, it employs an arrangement of metal wires, which
are strained from the object plane to the image plane and yields transfer of
the near-field with highly-to-moderately sub-wavelength spatial resolution.
Owing to strongly sub-wavelength confinement of the plasmonic modes on a

U
metal wire, each wire serves as a separate plasmonic waveguide and forms a
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separate pixel of the image. In Ref. [172], the magnifying hyperlens based
on WM has been proposed for the broadband THz imaging. In principle,
such a lens-less device for image formation could serve as an endoscope for
THz imaging of hard-to-access tissues. However, the problems of the WM
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handling and improving the speed and accuracy of the THz image detection
should be solved before applying the WM-based imaging in THz biomedicine.
Thereby, besides the conventional lens-based approaches of THz spec-
D

troscopy and imaging, which are characterized with diffraction-limited spa-

tial resolution, numerous sub-wavelength THz imaging modalities have been
TE

recently developed to overcome the diffraction limit. The methods of THz

SIL imaging and Tera-jet imaging, holography and synthetic aperture imag-
ing, near-field scanning-probe microscopy, superlens-based imaging and WM-
EP

based imaging have a potential to bring the spatial resolution of THz spec-
troscopy and imaging to the scales ranging from hundreds microns to tens
nanometers, which span the scale of different structural components of tis-
sues, including, the microfibrils, cells and cell organelles.
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Nevertheless, numerous research and engineering problems for adaptation

of the THz imaging modalities to study biological samples in vitro and in
AC

vivo should be solved before their widespread use in biomedical applications.

These problem include the use biocompatible materials, reduction of the size
and cost of spectroscopic and imaging systems, impovement of the accuracy
and speed of measurements.

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4.4 Microstructure waveguides for terahertz radiation

delivery to internal organs

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Delivering THz-waves to/from tissues that are hard to access and in-
ternal organs still remains a challenging problem owing to the lack of THz
waveguides and endoscopes. In Fig. 22, we show a typical scheme of the

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waveguide-enabled THz measurements of tissues. Along with low disper-
sion and propagation losses in a broad spectral range, the waveguides for
THz biomedicine should be fabricated from biocompatible materials, which

SC
features high chemical resistance and inertness to the human body fluids,
and possess a small cross-section. Furthermore, modern technologies for tis-
sue diagnosis and treatment combined in a single instrument and exploiting
electromagnetic waves of various spectral ranges [173,174], require the waveg-

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uides capable for multispectral operation. In this section, we describe recent
developments of the THz waveguides from the viewpoint of their applicability
AN
in the THz biomedical spectroscopy and imaging.
There have been many waveguides developed during the past decades.
The first type of the THz waveguides is based on the hollow metal tubes [175]
M
or tubes with an inner thin dielectric coating [176]. These waveguides show
small propagation and low-to-moderate bending losses [177]. However, they
feature significant modal dispersion in case of a small core size or multi-
D

mode guidance regime for a large core size. These factors limit reliability of
the tube-based waveguides in broadband THz sensing technologies (i.e., THz
TE
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Figure 22: A scheme of the THz waveguide use for measuring the hardly-
accessible biological tissues. (Courtesy of K.I. Zaytsev & M.M. Nazarov)

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pulse spectroscopy, imaging and time-of-flight tomography), which modern

biomedical applications of THz technology rely on.
The second type of THz waveguides is based on the microstructured,

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sub-wavelength, porous and multichannel [178], or antiresonant [179] and
photonic crystal [180] waveguides. Recently, microstructured polymer waveg-
uides were produced using several techniques, including drilling and draw-

RI
ing [181], stacking and drawing [182, 183], and 3D printing [184, 185]. By
optimizing the cross-section geometry and minimizing electromagnetic wave
– material interactions, the microstructured polymer waveguides allow for

SC
both managing the waveguide dispersion and reducing the propagation loss.
Being fabricated from transparent and chemically-resistant polymers, such
as polyethylene, polypropylene, Topas, Teflon, the microstructures waveg-
uides allow for use in numerous problems of THz biomedicine. For instance,

U
in Ref. [186], a dielectric pipe waveguide was demonstrated as a refractive
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index sensor for powder and liquid-vapor sensing.
Along with the discussed polymer waveguides, microstructured THz waveg-
uides were recently fabricated based on sapphire shaped crystals [187–189].
These waveguides combine the discussed advantages of electromagnetic waveg-
M
uidence in dielectric microstructures with the unique physical properties
of sapphire [190, 191] – i.e., high refractive index and relatively low THz
wave absorption, high hardness and mechanical strength, impressive melting
D

point and chemical resistance (even when operating at high temperatures

and pressures). In contrast to polymer waveguides and fibers, the sapphire
TE

shaped crystals are non-flexible. Thus, this type of waveguides does not pos-
sess bending losses and makes possible performing quantitative remote spec-
troscopy of tissues that are hard to access and internal organs, when straight
EP

endoscopes are applicable, for example, in intraoperative diagnosis of brain

malignancies [192]. Sapphire remains transparent at a number of spectral
ranges, including the ultraviolet, visual, near- and middle-infrared, THz and
millimeter-wave bands. This makes the sapphire waveguides attractive for a
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multispectral sensing and exposure [193].

In Fig. 23, we show representative examples of the THz microstructure
AC

dielectric waveguides, where panels (a) and (b) illustrate images of flexible
polymer waveguide with hollow-core and capillary cladding, and its cross-
section [183], while panels (c) and (d) demonstrate images of non-flexible
150-mm-length waveguide, based on sapphire shaped crystal, and its cross-
section, respectively [187].
When high spatial resolution is needed in spectroscopy and imaging of

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tissues, plasmonic waveguides might be preferable. This type of waveguides

is based on a single or dual metal wires and ribbons, or arrays of metal wires
and ribbons suspended in air [194–196]. Thanks to the guidance in air, these

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waveguides provide both low dispersion and propagation loss in a wide spec-
tral range. At the same time, the majority of plasmonic waveguides suffer
from low coupling efficiency and handling difficulties [197]. In Ref. [198], at-

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tempts to mitigate these difficulties by surrounding the plasmonic waveguide
with porous dielectric support have been applied, but the resultant waveg-
uides have lost their attractive small dispersion and propagation losses. Thus,

SC
the problems of electromagnetic wave coupling to plasmonic waveguides and
their handling are still unsolved, which makes them sub-optimal for THz
biosensing.
The step-index dielectric waveguides have been fabricated of polystyrene

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[199], bulk crystalline media [200] or crystalline foam [201]. Such waveguides
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seem to be attractive due to biocompatibility and, consequently, an ability
to be used in THz biosensing. At the same time, the cylindrical geometry of
the waveguide cross-section does not have any potential for reduction of the
THz wave losses and dispersion; therefore, it does not allow for using these
M
waveguides in a broadband THz spectroscopy and imaging.
By considering the described trends in the area of the THz waveguides’
D
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(d) 5 mm
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(b) 1 mm
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(a) (c)

Figure 23: Representative examples of microstructure dielectric waveguides

AC

for THz electromagnetic waves: (a) and (b) flexible polymer waveguide fea-
turing hollow-core and capillary cladding [183], and its cross-section, respec-
tively; (c) and (d) non-flexible 150-mm-length waveguide, based on sapphire
shaped crystal [187], and its cross-section, respectively. (Courtesy of K.I. Za-
ytsev & M.M. Nazarov)

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developments from the viewpoint of their optical performance and biocom-

patibility, we can conclude that the microstructure dielectric waveguides and
tubes with inner dielectric coating seem to be promising instruments of the

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THz biomedicine. These kinds of waveguides allow transmitting a broad-
band THz pulses to/from the object of interest in a spectral band of about
0.3 to 2.5 THz (this band is extensively used in THz medical diagnosis)

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over the length of tens of centimeters with the small dispersion of about
0.1...1.0 ps/(THz·cm) and the low propagation losses of about 1...10 dB/m.
Unfortunately, up to now, it is not possible to combine all required prop-

SC
erties in the particular THz waveguide. It is always a compromise between
transversal dimensions and lossess, single mode regime and dispersion, flex-
ibility and bandwidth. Thereby, novel technologies and new materials are
required to solve the demanding problems of THz waveguiding. Numerous

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research and engineering efforts are needed to bring these technologies into
clinical practice. AN
5 Interaction of terahertz radiation with free
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water, biological water solutions, and tis-
sue water
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5.1 Introduction
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In this section, we start with a discussion of THz spectroscopy methods,

which are widely applied for characterization of water and water solutions.
Then, we consider the THz dielectric response of free water, water solutions
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of biological molecules (i.e., sugars and proteins), blood and its components.
We demonstrate changes in the THz complex dielectric permittivity of water,
water solutions and biological tissues in cooled and frozen states. Finally,
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based on the presented THz dielectric spectra of water and water solutions,
we discuss related prospective applications of THz science and technology in
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biology and medicine.

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5.2 Methods of solution measurements

5.2.1 Transmission-mode measurements

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The most widely-exploited method of solution measurements using THz
spectroscopy implies studying the THz-wave transmission through a thin
analyte layer formed inside a cuvette [202, 203]. Usually, the thickness of

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this layer is about 50–100 µm, which is smaller than the electromagnetic
wavelength and is comparable with the depth of THz wave penetration into a
solution. In such measurements, even a small uncertainty of the analyte layer

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thickness might cause a noticeable error in the reconstructed THz dielectric
response. Furthermore, the physical properties of a solution may be modified
in the vicinity of interfaces between the cuvette windows and the analyte,

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as compared to the properties of a bulk liquid, thus, leading to additional
artifacts in the measured data [204].
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Hence, a thick cuvette is preferable, as it gives considerable advantages
in low frequency part of the THz spectra, where water transmission is not so
small. For example, in Ref. [24], the low-frequency THz-TDS equipped with
a multi-dipole emitting antenna featuring the average power of 10 µW, was
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applied for spectroscopy of water, providing an impressive spectral operating
range with the high-frequency cut-off at about 1.0 THz even when a 0.5-mm-
thick water layer was characterized. This spectrometer yields measurements
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of a 103 -times decrease in the THz field amplitude and, thus, a 106 -times
decrease in the THz beam power. Furthermore, to achieve a stable and
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reproducible result of at low frequencies (∼ 0.07 THz), a rather large time-

domain window was used for the THz-TDS waveform detection. In this work,
processing of the THz-TDS data was performed in a manner similar to that
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mantioned above (see section 3).

5.2.2 Reflection-mode measurements

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For high THz frequencies, the ATR measurement approach is preferable,

since it does not suffer from strong water absorption and implies measure-
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ments of a bulk sample; thus, eliminating an error of measurements associated

with an uncertainty in the analyte layer thickness [26,205,206]. Typically, an
ATR signal is sufficient to measure a reflection spectrum of a solution in the
range of 0.25–3.50 THz, while it is important to fix the polarization normal
to the prism base – i.e., so-called p-polarization. The reflection from the
empty prism is used as the reference signal Er (ω), while the reflection from

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the prism with a solution poured onto its top surface is used as a sample sig-
nal Es (ω). One should notice that the thickness of the analyte layer should
be larger than the depth of THz-wave penetration into a solution – namely,

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larger than 1 mm for low-frequencies (∼ 0.1 THz). The phase spectra are
calculated as ϕ (ω) = −arg [Es (ω) /Er (ω)]. As compared with the conven-
tional THz-TDS reflection-mode measurements; in the ATR approach, the

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modulus of reflectivity |Rp (ω)| = |Es (ω)| / |Er (ω)| is relatively high (up to
70 %); thus, providing higher sensitivity. The complex ATR spectra Rp (ω)
allows one to extract the complex dielectric permittivity of the sample ε (ω)

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as follows
r
1+Rp (ω) 2 1+Rp (ω) 4 1+Rp (ω) 2
    
1−Rp (ω)
± 1−Rp (ω)
− 2 sin2 (2θ) 1−Rp (ω)
ε(ω) = nprism 2 , (48)

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2 cos2 (θ)
where θ = 57◦ is an incident angle inside the prism, nprism is a refractive index
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of the prism material (nprism ' 3.42 for the HRFZ-Si prism). Before studying
an unknown solution, one should confirm the applicability of parameters used
in Eq. (48) by measuring the well-described distilled water; the exact values
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of incident angle θ, refractive index of the prism, reliable frequency range,
sufficient thickness of the analyte layer should be determined.
One should choose an appropriate experimental method for each part
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of the THz spectral range: transmission-mode measurements for the low-

frequencies; ATR measurements for the high frequencies. Actually, these
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methods have considerable overlap of the spectral operation range; thus, one
can combine the results obtained by these methods in order to achieve a
broader spectral operation range and more reliable data for water solution
spectroscopy [24, 206].
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5.3 Approaches for description of dielectric response

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of solutions in the terahertz range

Once the water spectrum is precisely measured and described (see Tab. 1),
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one can proceed to water solution analysis. For good accuracy of measure-
ments, it is not reasonable to vary all the parameters of the solution model,
defined by Eq. (18). In the first approximation, it is useful to determine the
main process in Eq. (18) and the corresponding physical parameter, which
can describe the changes of dielectric spectrum. Further, it is necessary to
get the dependence of this parameter on the concentration that will provide

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preliminary information about the physical and chemical processes in a so-

lution. In particular, the bend of the concentration dependence determines
the size of the hydration shell; at the bend point, the hydrate shells begin to

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overlap with each other.
In Fig. 24, schematics of the solution composition is presented; each
molecule in a solution is surrounded by a bound water or hydration shell.

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Slow relaxation spectra of bound water are shifted to much lower frequen-
cies [207]; thus, in GHz and THz range bound water has smaller absorption
and dispersion. That is the main reason of solution spectra changes.

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In general, we should use the effective medium approximation for the total
spectrum; in a more simplified case, we should take into account the volume
fractions of the three solution components with different spectral responses:

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1. the molecules of the solute (they do not experience strong relaxation
process, accompanied by a sharp increase of dielectric permittivity at
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low frequencies); they simply displace part of the water volume;

2. the bound water forming the hydrated shell around each molecule of
the solute; such water molecules have a smaller relaxation time τ1 ;
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3. the remaining free water with the initial parameters – its contribution
to the THz spectrum of a solution dominates even at high solution
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Figure 24: Free and bound water in the bio-solution. (Courtesy of

M.M. Nazarov)

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concentration.

For this approach, described in Ref. [23], we have

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Vsolute Vhydration
   
αtotal = αsolute + αhydration
Vtotal Vtotal
(49)
Vtotal − Vhydration − Vsolute

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+ αwater ,
Vtotal
where Vtotal , Vsolute , and Vhydration are the volume of the solution, the so-

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lute, and the hydration water, respectively; while αtotal , α√solute , αhydration , and
αwater are related THz absorption coefficients (α = 2ω/c ε00 ). It is more rig-
orous to manipulate with ε in a similar way [22], or even apply effective

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medium approximation. Equation (49) yields prediction of small changes in
solution spectra; however, it can be further simplified.
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In order to qualitatively understand the changes in the THz spectra of
aqueous solutions, the following assumptions can be introduced:

1. ε00solute  ε00water – the role of the solute is mainly the replacement of

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strongly absorbing water from the volume;

2. τ1,hydration  τ1,water – the response of the bound water is shifted to the

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MHz-GHz frequency range, while, in the THz range, the contribution

of τslow,hydration is practically very small.
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If both the 1st and the 2nd assumptions are satisfied, Eq. (49) is simplified
to
ε00total (Csolute ) ≈ (1 − Chydration − Csolute ) ε00water , (50)
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where C is the corresponding volume concentration, Csolute = Vsolute /Vtotal ;

Csolute is known from the density of the solution [208]. It is possible to cal-
culate the Chydration from Eq. (50) and to determine the size of the hydration
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shell.
If the 1st assumption is not valid, but the estimate for ε00solute is known [205],
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then the difference between the calculated curve (with Chydration = 0) and
the experimental curve provides the reconstruction of Chydration without any
simplifications [23, 205].
If we assume that the ”fast” relaxation process in Eq. (3) is approximately
the same for the hydration (bound) and the free water, a dependence of the

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amplitude and the shape of a solution spectrum on the concentration C can

be described using a single parameter ∆εslow = ∆ε1 [24, 206]

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∆εslow (Csolute ) = ∆εslow (0) (1 − Csolute (1 + x)) , (51)

where x is a specific constant, defined for the partiular solution and account-
ing for the size of the hydration shell. The applicability of this approach is

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demonstrated in Fig. 25, where the rest terms in Eq. (3) (fast relaxation and
overdamped oscillations) are taken from the pure water. Such a simplified
approach, implying variation of a single parameter ∆εslow (Csolute ), describes

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well the experimental spectra of aqueous solutions (particularly, of sugars
and proteins) in the frequency range of 0.3 to 2.5 THz, which is important
for practical applications of THz-TDS systems [206]. In any case, in this THz

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band, 80 % of the spectral response is determined by the slow relaxation and
its parameters (∆εslow , τslow , α), while contributions of the remaining pro-
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cesses in Eq. (3) are relatively small and weakly depend on the concentration.
For example, in Ref. [67] the parameters of all processes, except the slow one,
undergo less than 10% change for the saturated solution of disacharides.
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5.4 Complex dielectric permittivity of sugar solutions
in the terahertz range
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Since there are practically no standards and calibrated instruments in

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the THz range, before studying a new system, it is necessary to achieve an

agreement of the measured data with the well-known literature results on
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Figure 25: Bulk water relaxation strength as a function of disaccharide con-

centration. (Reproduced from the Ref. [22], with the permission of AIP
Publishing)

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the THz dielectric response of some ”reference” solute, for example, glucose
[24, 205, 208].
In Ref. [205], a spectrum of bound water was extracted for the glucose

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solution with C = 1.5 M at 27 ◦ C; its slow relaxation time τδ = 21 ps and
the frequency-independent spectrum of the glucose molecules εsolute = 3.0
were observed. The total spectrum is well described by the sum of three

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terms, corresponding to the free water (with the magnitude ∆εslow = 40),
the bound water (with the magnitude ∆εslow = 25) and the solute. The same
solution was measured in Ref. [208] using the dielectric spectroscopy method;

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its dielectric response is well described in the range from MHz to 0.1 THz
using only the ”slow” term in Eq. (3) in the Cole-Cole form: for C = 1.5 M
and 25 ◦ C, the Cole-Cole term parameters are ε∞ = 2.85, ∆εslow = 72,
τslow = 13 ps, and α = 0.87. In Ref. [24], for the THz-TDS method and the

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0.07–2.50-THz-range, similar glucose solution is well described by Eq. (18)
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with all processes parameters the same as for water, except ∆εslow , which has
a value of ∆εslow = 53 for C = 1.5 M, and of ∆εslow = 29 for C = 4.7 M; thus,
conforming applicability of Eq. (51). Thereby, different models lead to almost
equal values of εtotal (defined within the limits of measurement errors) in the
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frequency range where the models overlap. For example, for the saturated
glucose solution of C = 4.7 M, in Ref. [208], we could find a complex dielectric
permitivity of εtotal = 6.6 − 5.0i at 80 GHz and 25 ◦ C, while in Ref. [24], we
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obtain εtotal = 6.6 − 6.0i for the same concentration and frequency, but for
the slightly lower temperature of 22 ◦ C. For a more realistic concentration
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C = 1.5 M, in Ref. [24], we obtain εtotal = 7.7 − 11.0i at 22 ◦ C, while in

Ref. [207], we observed εtotal = 8.0 − 11.0i at the slightly higher temperature
of 27 ◦ C. For the same concentration and higher frequency of 2.0 THz, we
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observe quite similar values of εtotal = 3.9 − 1.5i and εtotal = 3.7 − 1.5i from
the Refs. [24] and [207], respectively.
The intrinsic (intramolecular) resonances in sugar solutions appears only
at frequencies above 5.0–7.0 THz; for example, a small peak at 8.5 THz is
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assigned as the intramolecular vibration mode of trehalose in Ref. [22].

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5.5 Complex dielectric permittivity of protein solu-

tions in the terahertz range
For protein solutions, the most studied sample is BSA [23, 202, 209, 209];
thus, it can be used as an etalon. In our research, we prolong the systematical

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studies of BSA solutions to the case of ”low” concentration and increased

frequency range – i.e., we shift the low-frequency limit down to 0.07 THz.
In Fig. 26, we demonstrate the frequency-dependent real ε0 and imaginary

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ε00 parts of complex dielectric permittivity for the aqueous solution of BSA
with the concentration of 100 mg/ml. Despite some ”resonance” frequencies
of protein solution, are regularly reported in the THz range, for a simple

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aqueous solutions of BSA, the THz spectra are featureless. Only in recent
Refs. [210] , the authors reveal the presence of resonances in the THz spectra
of a specially-prepared and laser-excited BSA aquas solution.

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Dielectric spectroscopy has made a significant contribution to the study of

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Dielectric function

20 -Im( ) water

-Im( ) BSA solution

Re( ) water
15

10
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5
M
0.1 1

Frequency (THz)
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Figure 26: Real ε0 and imaginary ε00 parts of the complex dielectric permittiv-
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ity for water and a 100 mg/ml aqueous solution of BSA at 21 ◦ C. (Courtesy
of M.M. Nazarov)
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Figure 27: Dielectric spectra of aqueous protein solutions. (Reprinted from

the Ref. [211], copyright (2012) with permission from Elsevier)

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protein solutions [13]. From Fig. 27, we observe that three dispersion regions
are commonly found in a protein solution – β, γ, and σ-relaxations. The
β-relaxation, occurring in the frequency range around 10 MHz, and the γ-

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relaxation, centered near 20 GHz at room temperature, can be attributed to
the rotation of the polar protein molecules in their aqueous medium and the
reorientational motion of the free water molecules, respectively [211]. Using

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a Debye function for the β process and a Cole-Cole term for the γ-relaxation,
the complete broadband spectra can be well fitted. However, dielectric spec-
troscopy does not provide information on the variety of interactions between

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water and protein in its hydration cell.
When studying protein solutions in the THz range, several research di-
rections can be distinguished.

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1. Study of the collective motion of water molecules around protein molecules.
M. Havenith’s group has pioneered THz absorption spectroscopy as a
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tool to probe collective sub-ps hydration dynamics of biomolecules us-
ing the p-Ge laser in the frequency range of 2.4 to 2.7 THz [23, 212,
213]. Fundamental results were obtained on the interaction of water
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molecules with a dissolved protein; it was established that this influ-
ence is mutual. It is shown that there several layers of the hydration
shell exists, and the relaxation times near the protein molecules are
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much slower than that in bulk water.

2. The study of protein solutions of different concentrations by THz spec-

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troscopy was carried out by many groups [59, 202, 206, 209, 214, 215].
The molar absorption lysozyme [214] and BSA [215] were investigated
in the range of 0.08–3.72 THz. It was shown that molar absorption of
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lysozyme increases with frequency and saturates above 2.0 THz. At

the same time, the molar absorption of BSA increases monotonically
and does not significantly depend on concentration, except for very di-
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luted solutions, for which an uncertain growth of molar extinction was

observed [215]. An increased absorption of diluted protein solutions
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relative to water uptake was found in Ref. [59, 202].

In the case of diluted protein solutions, the THz range contain a high-
frequency tail of the γ-relaxation spectrum (slow relaxation process), which
is described by the Cole-Cole model. With increasing protein concentra-
tion, a peak in the ε00 (ν) dependence broadens (α < 1), while the relaxation

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(0)
0.1 THz measurment

1
1.00
3.0 THz measurment

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(C)/
1
0.95

0.90

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0.85

0 20 40 60 80 100

BSA concentration, mg/ml

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Figure 28: A nonlinear dependence of ∆εslow on the concentration of a solute
BSA in water, where a bend near the concentration of about 30 mg/ml is

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observed. (Courtesy of M.M. Nazarov)

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time τslow increases [216]. A more-faster-than-linear dependence on the vol-
ume fraction of protein, along with a decrease in the amplitude of ∆εslow ,
may originate from changes in the structure of free water near the protein,
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which should change the shape of ε (ν) spectrum at low frequencies [59].
The dependence of εslow on the protein concentration is nonlinear; thus, from
the Fig. 28, one could notice the line bend near the BSA concentration of
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30 mg/ml [217].
Since the main Debye relaxation peak is centered near the few tens of
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GHz, the highest sensitivity to the protein concentration in a solution is

observed in this range. In Ref. [218], it was reported that the use of Gunn
diode with the output frequency of 60 GHz allows for reliable determination
of 1 wt% BSA in a solution.
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5.6 Complex dielectric permittivity of blood and its

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components in the terahertz range

Blood consists of plasma, which constitutes 55–60% of the whole blood
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volume, and cellular elements. There are three main types of blood cells: red
blood cells (RBCs) or erythrocytes, white blood cells (leukocytes), and blood
platelets (platelets). Blood plasma contains 90% of water, about 6.6–8.5%
of proteins (most of which are albumins) and other organic and mineral
compounds (including glucose and salts), which are intermediate or final
products of metabolism, transferred between organs. As it was mentioned

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Figure 29: Optical constants of water, plasma, blood, and RBCs: (a) refrac-
tive index and (b) absorption coefficients. The lines and error bars represent
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the mean values and standard deviations of the optical constants of three
samples, each obtained from different rats. (Reprinted from the Ref. [220])
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above, all these components affect the water structure, which constitutes the
main part of the blood.
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The whole blood and its components were studied using the THz-TDS
method in a number of papers [206, 219–222]. From Fig. 29, we could notice
that the THz absorption and reflection spectra of the blood and its compo-
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nents have no sharp spectral features [220]. The shape of the THz spectra of
the blood is close to that of water; for its analysis, it is possible to use the
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double-Debye model described above (see section 2) [206, 219]. The param-
eters of ”slow” relaxation process were determined for blood components of
healthy man: the relaxation time τslow for the whole blood, blood plasma and
RBCs are 14.4, 8.0 and 410.8 ps, respectively; while, for the blood plasma
and water, τslow is similar [219].
The absorption coefficient of blood decreases linearly with an increasing

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in RBC concentration [220, 222]. A pilot clinical study of ex vivo fresh hu-
man whole blood of 28 patients before cardiac catheterization showed that
triglycerides and the number of RBC were two dominant factors possessing

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significant negative correlation to the sub-THz absorption coefficients [222].
The authors concluded that concentration of RBCs and triglycerides needs
to be limited within a narrow range for future THz investigation in human

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whole blood.
The absorption coefficient and the refractive index of diabetic rat blood
plasma are small compared to those of water and plasma of healthy rats [221].

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The diabetic rats have a reliably high level of corticosteroid hormones in
blood and adrenal glands, a large weight index of kidney, and a higher level
of glucose in blood in comparison with healthy rats [221]. Assuming that
the observed spectral changes are due to changes in the state of water in

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blood plasma, we have selected one of the parameters of the Debye model of
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the aqueous solution, ∆εslow /τslow , leading to the spectral features observed
in the experiment. This change in the response of bound water can be the
reason of the observed changes with an increase of glucose concentration in
blood. We have demonstrated that when the concentration of glucose in
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blood rises to 24 mM (in rats with diabetes), ∆εslow / τslow ratio decreases
in 1.2 times [206].
A reduction in the absorption coefficient of blood plasma from mice with
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experimental oncology (Ehrlich carcinoma), as compared to that of healthy

mice, was demonstrated in Ref. [223]. The authors concluded that such com-
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ponents of whole blood as RBCs and platelets make the major contribution
to the blood absorption in the THz range. In this relation, the changes in
the composition of blood plasma cannot be reliably detected in the whole
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blood using the methods of THz spectroscopy. The blood plasma is the most
promising object of study, since the change of composition caused by patho-
logical processes can considerably affect the optical properties of the blood
plasma of humans and animals in the THz range [221, 223].
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Our approach to describing the THz dielectric response of biological so-

lutions is as follows.
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1. The known parameters of water model and refining of the range of

reasonable values for each of them at room temperature were system-
atically analyzed.
2. The reference parameters of each relaxation process from a correspond-
ing frequency range and appropriate method of measurements were

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taken.
3. The ”slow” Debye relaxation was assumed as representing the only

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significant process, while other terms introduce negligible contribution
to changes in the dielectric permittivity of aqueous solutions in the THz
range. Thus, for approximation of a complex dielectric permittivity,
we introduce certain constraints on each parameter, proceeding from

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the data taken from the frequency range appropriate to each of the
processes. To analyze the data in the range of 0.07 to 3.2 THz at

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an ambient temperature (20-25 ◦ C), we select the following parameter
ranges, which do not contradict the most reliable published data: ε∞ =
2.1 to 2.7, ∆εslow = 72 to 75, τslow = 8.3 to 9.5 ps, ∆εfast = 1.4 to 2.1,
τfast = 130 to 360 fs, εs = 77.0 to 80.2. Then, we obtain the relevant set

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of parameters, which accurately describe both our experimental results
and data published by other authors.
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We reviewed recent attempts to understand the physical processes un-
derlying the THz complex dielectric permittivity of biological or medical
solutions. The informativeness of THz-TDS data for biomedical solutions
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is still remains a big question, since any sample containing water does not
show distinct spectral features on THz frequencies. At the same time, the
THz waves allows for determining accurately the concentration of water, and
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can be used for differentiation between its free and bound states. But for
the latter application, the sensitivity should be better in GHz range, since
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the absorption peak (slow Debye relaxation) of water is in the region of 20–
30 GHz. Near this relaxation peak, the difference between free and bound
states are the most pronounced. In ”classical” range of 0.3 to 3.0 THz, we
study only the far tail of this wide peak, but with phase information and
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temporal resolution. Other two known processes in water (fast relaxation

around 1.0 THz and Lorenzian overdamped resonance at 5.2 THz) do not
show enough sensitivity to solution or tissue states of water. Anyway, for the
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accurate analysis of liquids, one needs accurate complex (phase sensitive)

measurements from 1.0–2.0 THz to as low frequencies as possible.
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5.7 Terahertz spectroscopy of ice, biomolecules and

tissues in cooled and frozen states
Since THz waves are sensitive to water content in tissues and are capable
for differentiation between its free or bound states, water could be used as

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Figure 30: Refractive index and absorption coefficient of water in the THz
range. Lowering the temperature from 0 ◦ C (liquid water) to -5 ◦ C (ice)

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produces a huge decrease in the absorption coefficient. (Reprinted from the
Ref. [225]) AN
an endogenous marker in label-free diagnosis of numerous pathologies, in-
cluding malignancies in different localisations [224, 225]. Meanwhile, such a
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strong THz wave sensitivity to water content in tissues, caused by significant
THz wave absorption by water, presents drawbacks when it is used to study
either biological samples immersed in water or the depth regions of hydrated
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tissues. This problem can be alleviated significantly by freezing samples in

or with water because the absorption of THz waves by water ice drops to
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approximately one-twentieth the absorption rate of liquid water, when the

temperature is lowered from 0 ◦ C to the cooled state of -5 ◦ C, as shown
in Fig. 30 [225]. Fast freezing a sample has been reported to leave both
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biological behavior and cellular or tissue structures unaltered [226].

One study, which observed resonant THz fingerprints in cancer DNA, of-
fers a good example of studying biological molecules in a cooled state [227].
A previous study has reported that the DNA of cancer cells show a chemical
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change called methylation, which rearranges the 5-methylcytidine distribu-

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tion [228]. In this process 20 -deoxycytidine is altered to 5-methylcytidine in

CpG islands that have molecular resonances in the THz region. The mea-
surement of the methylation in cancer DNA requires freezing the sample to
-20 ◦ C. If the sample had not been cooled to this temperature, most of the
probing THz signal would have been attenuated by the liquid water in re-
laxational mode. In Fig. 31, we show the molecular resonance observed at

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Figure 31: Resonance peaks of the THz absorption coefficients of two control
samples of DNA – kidney-cell line (293T) and artificially methylated 293T

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(M-293T), and five types of cancer DNA – prostate cancer (PC3), skin can-
cer (A431), lung cancer (A549), breast cancer (MCF-7), and gastric cancer
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(SNU-1], originating from methylation measured at -20 ◦ C. The effects of ice
and common molecules were removed using the baseline correction method
(Reprinted from the Ref. [227])
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approximately 1.67 THz for five types of DNA extracted from cancer cell
lines [227]. Although the degrees of methylation differed among the types of
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cells, as shown by the varying amplitudes of the absorption peaks, the peak
positions occurred at the same frequency. This alignment implies that all
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types of cancers show similar chemical changes through methylation, which

yields identical resonant energies. Researchers believe that this effect origi-
nates from the collective motion of the methyl groups interacting with water
molecules.
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THz spectroscopy can serve as a novel technique that complements con-

ventional medical imaging modalities, such as magnetic resonance imaging
and X-ray computed tomography, for providing diagnostic cancer imaging.
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However, this THz technique is valid only to image the superficial parts of
a tissue sample because the penetration depth of THz waves is limited to
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a few hundred of micrometers in wet biomedical tissues either ex vivo or in

vivo. However, the penetration depth can be increased several times sim-
ply by freezing the tissue. In Fig. 32, we demonstrate this approach with
a human oral squamous cell carcinoma sample. The cancerous region was
1.3 mm below the oral tissue and was not detectable in wet conditions at
room temperature. The probing THz waveform, however, showed a second

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Figure 32: Histological image of the cross section of an oral tissue sample
and the reflected terahertz time-domain waveforms from the tissue (a), at

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room temperature (b) and at -20 ◦ C (c). The cancerous region was located
1.3 mm below the tissue surface. The tumor deep inside the tissue was
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identified by the second reflected pulse (black arrow). This identification
was made possible by freezing the tissue to enhance the waves’ penetration
depth. (Reprinted with permission from the Ref. [229], Optical Society of
America)
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peak reflected by the tumor region after the penetration depth had been
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improved by the tissue freezing to -20 ◦ C [229]. This freezing technique for
tissue imaging also was applied to differentiate healthy and metastatic lymph
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nodes [230]. The THz signals reflected by both types of lymph nodes could
not be distinguished at room temperature because of the high attenuation
contributed by the liquid water in the tissues. In contrast, a signal gap be-
tween the healthy and metastatic lymph nodes was detected in the frozen
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samples.
Thereby, the THz technique is valid only to image the superficial regions
of a tissue sample because the penetration depth of THz waves is limited
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to a few hundred micrometers in hydrated tissues either ex vivo or in vivo.

However, the penetration depth can be increased several times simply by
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freezing the tissue.

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6 Terahertz spectroscopy and imaging of ma-

lignant tumors

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6.1 Introduction
Over the past two decades, considerable efforts have been dedicated to

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the development of the clinical applications of THz technologies [231]. Even
though remitted THz signals do not exhibit resonant features, statistically
significant differences in THz properties of healthy and malignant tissues

SC
have been reported for several cancer types [232]. It has been suggested
that the differences between dielectric permittivity of healthy and malignant
tissues are primarily determined by water content. Similarly to water, the

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THz dielectric responses of tissues are described by relaxation models of
complex dielectric permittivity [233], such as Debye [234] or Cole-Cole [52].
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According to these models, the real part of permittivity decays monotonously
with frequency increase, and the imaginary part presents broad absorption
bands. High water content in tissues leads to strong absorption and shallow,
on the order of several hundred microns, penetration of the THz radiation
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into tissues. Thus, only the reflection-mode measurements could be utilized
for the THz detection of malignancies in vivo [235].
It has been suggested that the contrast in the THz images between
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healthy and malignant tissues is determined by the differences in morphol-

ogy [173, 236–238] and water content [173, 236–243] in tissues. Abundant
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vascularization and edema of cancerous tissues may yield higher water con-
tent, as well as variability of the THz refractive index and absorption coef-
ficient [244]. Multiple scattering of THz waves on tissue inhomogeneities is
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often neglected under an assumption of low elastic scattering. This approach

is valid when the size of scatterers d is small compared to the THz wavelength
λ (see section 4.3, Fig. 19). However, for some tissues, such as skin, the size
of the tissue structures, i.e., hair, hair follicles, sebaceous glands, and other
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appendages, is on the order of millimeters. Moreover, macroscopic varia-

tions of tissue refractive index may results in significant multiple scattering,
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especially for the frequencies above 1.0 THz [245–248].

The majority of THz systems for malignant tissue detection employ THz-
TDS and THz pulsed imaging (see section 3), which often requires point-by-
point scanning of a sample with a focused beam and further processing of
3D data sets [235]. THz-TDS measurements enable simultaneous detection

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of both frequency-dependent amplitude and phase of the THz waveform in

a broad spectral range. The results of such measurements yield the elec-
tromagnetic properties of tissues and may be processed in either the time

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or frequency domain. The instrumentation for THz-TDS and THz pulsed
imaging, as well as the data processing algorithms for accurate characteri-
zation of tissues are well-developed [235]. Several portable, handheld and

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ergonomic devices were implemented for clinical translational research [249].
Typical THz-TDS or THz pulsed imagers yield the effective tissue response
averaged over the area of the THz beam (see section 4.3, Fig. 19). Along with

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THz-TDS and THz pulsed imaging, numerous approaches relying on the use
of CW THz radiation are extensively developed and being increasingly used
in THz biomedical applications [238,250], as single frequency imaging device
costs a fraction of the price of a THz-TDS and a THz pulsed imaging system

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and is much easier to implement.
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Considering strong attenuation of THz waves by water, only superficial
tissue imaging and spectroscopy can be translated into clinically relevant
applications. So far, there are three major thrusts:
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• noninvasive THz detection of cancers;

• intraoperative detection of cancers ex vivo and in vivo;

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• intrinsic contrast THz histopathology.

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6.2 Noninvasive terahertz diagnosis

Malignancies of the skin can be noninvasively studied using the reflection-
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mode THz pulse spectroscopy and imaging.

Skin is usually modeled as a multi-layer structure comprised of several
layers – i.e., the stratum corneum (the outermost layer of the epidermis, con-
sisting of dead cells), epidermis, dermis, and hypodermis (adipose tissue);
C

see Fig. 19. The THz dielectric response of healthy skin varies significantly
along the human body owing to both fluctuations of dielectric properties of
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tissues [98] and thicknesses of the skin layers [98, 251]. The depth of THz
wave penetration into the skin in vivo is limited with a few hundreds microns,
which typically allows for examination only the top layers of the skin – i.e.,
the stratum corneum and the epidermis. Furthermore, the thickness of the
stratum corneum is usually too small (compared to the THz wavelengths) to

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Figure 33: The results of the THz pulsed imaging of BCC in vivo: (a) a clin-
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ical photo of the BCC sample; (b) a THz parametric image of the BCC
sample, each pixel of which represents the minimal amplitude of the THz
pulse mint [E (t)] reflected from the sample of interest and shows surface fea-
M
tures of tissues; (c) a THz parametric image of BCC sample, each pixel of
which represents the normalized amplitude of the THz pulse for the time-
delay of t0 = 2.8 ps and reveals the depth features of tissues; (d),(e) results
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of histological examination of tissues. (Reprinted from the Ref. [240])

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have any measurable impact on the THz characterization of the skin; there-
fore, THz methods characterize mostly living epidermis of the human skin.
Statistical difference in THz dielectric properties and images of healthy and
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malignant tissues have been demonstrated for non-melanoma cancers of the

skin, such as basal cell carcinoma (BCC) [240, 241] and squamous cell car-
cinoma (SCC) [173]. Both in vitro and in vivo non-melanoma skin cancers
possess higher THz refractive index, absorption coefficient and dispersion
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compared to the healthy tissues [240, 241]; this leads to increased reflectiv-
ity and broadening of the THz pulses reflected from malignant tissues. In
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Fig. 33, we show a representative example of the THz pulse imaging of BCC
sample in vivo from the Ref. [240]: (a) stands for a clinical photograph of the
BCC sample; (b),(c) stand for THz parametric images of the BCC sample
revealing its surface and depth features, respectively; (d),(e) stand for results
of histological examination of tissues.

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3.5 3.5

3.0
3.0

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2.5

2.5
2.0

ε’’
ε’

1.5
2.0

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1.0
- Dysplastic nevus
1.5 - Ordinary nevus
0.5
- Healthy skin
(a) (b)

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1.0 0.0
0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
ν, THz ν, THz

Figure 34: THz dielectric response of ordinary and dysplastic skin nevi, and

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healthy skin in vivo: (a),(b) real ε0 and imaginary ε00 parts of the complex
dielectric permittivity, respectively. (Courtesy of K.I. Zaytsev)
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Either time-domain or frequency-domain data of THz-TDS and THz pulse
imaging, or the changes in the polarization of CW THz radiation could serve
M
as useful information for discriminating the non-melanoma cancers from the
normal skin. The THz methods could be used for preoperative delineation
of non-melanoma skin cancers.
D

Along with noninvasive diagnosis of non-melanoma skin cancers, the THz-

TDS and THz pulse imaging may be useful for differentiation between ordi-
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nary and dysplastic nevi of the skin [248, 252]. In Fig. 34, we show a typical
THz dielectric response of ordinary and dysplastic nevi, and healthy skin in
vivo: (a) and (b) show real ε0 and imaginary ε00 parts of the complex dielectric
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permittivity, respectively. The differences between the dielectric properties

of ordinary and dysplastic skin nevi can be clearly appreciated. They point
towards the feasibility of differentiating types of pigmented skin neoplasms.
As dysplastic nevi are considered to be precursors of melanoma, the dead-
C

liest form of skin cancer, the ability to detect them early is an important
healthcare problem [253,254]. Thus, the THz spectroscopy and imaging may
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become an effective tool for early noninvasive diagnosis of dysplastic skin

nevi and skin cancers in situ.

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Figure 35: THz pulsed imaging of freshly-excised healthy, dysplastic and can-
cerous tissues of the colon: (a) THz parametric image of tissues, each pixel of
which shows a minimal amplitude of the THz waveform mint [E (t)] reflected

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from the tissues; (b) results of histological examination of tissues overlapped
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with a visual image; (c) results of histological examination of tissue over-
lapped with its THz image. In panel (c), the regions A and B correspond
to normal tissues, while C and D stand for dysplastic and cancerous tissues,
respectively. The observed results of the THz pulsed imaging agree well with
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the data of histology. (Reprinted from the Ref. [255])
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6.3 Least-invasive terahertz diagnosis

Several reports of pilot trials have presented promising results and in-
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dicate the significant potential of THz spectroscopy and imaging for non-
invasive detection of hard-to-access internal organs. For example, Fig. 35,
demonstrates results of THz pulsed imaging of freshly-excised colon tissues
from the Ref. [255]. The images contain regions of healthy, dysplastic and
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cancerous colon. They clearly indicate an ability of the THz spectroscopy

and imaging to detect colon pathology. In other studies, the feasibility of
utilizing THz waves for detecting cancers and pre-cancerous conditions of
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oral mucosa [229], colon [255], and gastric [256] tissues are shown.
Further development and clinical translation of these methods hinge on
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the development of THz waveguides and endoscopes. Currently, THz wave

delivery to the internal organs presents a formidable problem. However, mod-
ern developments in the area of THz waveguides and endoscopes, discussed
in section 4.4 of this review paper, give hope that clinically viable prototypes
can be implemented in the near future.

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Figure 36: An ex vivo study of BCC, imaged immediately after excision
without immersion in any culture medium. The THz image (top panel) is
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oriented to the visible image (bottom panel). The character X and the white
dashed line in the THz image mark the location of the histological section of
tissue specimen, while the symbol ”*” in the visible image marks the tumor.
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(Reprinted from the Ref. [241] with permission from Elsevier)

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6.4 Intraoperative terahertz diagnosis

THz technologies show promise for the intraoperative label-free delin-
eation of skin, breast and brain tumors. The efforts to utilize THz radi-
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ation for intraoperative delineation of malignant tumors were pioneered in

2002 [241–243]. In particular, it has been demonstrated that THz radiation
provided reliable contrast of nonmelanoma skin cancers. Representative im-
C

ages of BCC from Ref. [241] are presented in Fig. 36. The authors suggested
that this contrast arises from the differences in water content between normal
AC

and pathological tissue. To extract tissue information and enhance the con-
trast of tumor image, they developed THz data processing techniques such
as time post pulse and integral image [236, 239–243]. They have shown that
the THz pulsed imaging could probe comparatively deep layers (∼ 1 mm)
of biological tissue by registration both amplitude and phase information of
the remitted wave.

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Work by other groups has confirmed these findings [236]. In particular,

it has been confirmed that nonmelanoma skin cancer lesions exhibit high
contrast in the THz spectral range. The images of a representative specimen

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with BCC from Ref. [236] are shown in Fig. 37. In the images, the black
solid arrow points toward the tumor area. In the THz image, the cancer is
characterized by higher power values. The margins of the tumor, outlined

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by the dermatopathologist, are shown in the histology image (Fig. 37 (b)).
A power value image at 0.47 THz (Fig. 37(a)) demonstrates that the shape
and location of the tumor correlate well with those in the histology image.

SC
However, the same study discovered that in some cases, THz images also
highlight normal tissue. For example, in Fig. 38, we present 0.47 THz power
value image, which highlights several regions. Their comparison to the re-
spective histopathology, shown in Fig. 38 (b), reveals that only one of them,

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outlined with the black line, was involved with cancer, whereas the other
ones were normal. AN
To remedy the problem, it was proposed in Ref. [173,236] to combine THz
imaging with other, higher resolution optical imaging modalities, such as, for
example, optical polarization imaging. The authors of the Refs. [173,236,237]
M
have demonstrated that THz and optical imaging yield complimentary infor-
mation. THz interrogation provides high contrast images of cancer, whereas
optical images deliver high resolution morphological information that allows
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for resolving small skin structures such as hair follicles, sebaceous glands,
collagen fibers; thus, eliminating false positives often occurring in THz imag-
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ing. Examples of optical images compared with both THz and histopathology
images of the same tissues are presented in Figs. 37 (c),(d) and 38 (c),(d).
Comparison of the high-contrast regions in the THz images to the optical im-
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ages, shown in Figs. 37 (c) and (d), demonstrates that they contain clearly
defined collagen, sebaceous glands, and hair follicles.
To facilitate the comparison, the authors presented magnified regions of
interest, shown in Fig. 39. These areas are indicated with black squares in
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histopathology (Fig. 38 (b)). Close inspection of the optical images was espe-
cially useful for the highlighted areas in Fig. 39 (e). They present the regions
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of subcutaneous fat. In the low magnification optical images in Figs. 38 (c)

and (d), these areas may be confused with cancer; however, in Figs. 39 (k)
and (l), we observe the islands of subcutaneous fat. This observation is con-
firmed by correlation with the histopathology (Fig. 39 (j)). The synergy
between THz and optical imaging positions by their optimal combination al-
lows for the intraoperative delineation of non-melanoma skin cancer margins.

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AN
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Figure 37: Images of a representative skin sample with BCC: (a) a THz
power value image at 0.47 THz, where TPI stands for THz pulsed imaging
D

technique; (b) a hematoxylin and eosin (H&E) stained histology image of a

5-µm-thick section of tissues, where the tumor region is outlined with the
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black solid line; (c) an optical polarized light image (PLI); (d) an optical
cross-polarized image (CPI). Solid arrow indicates tumor site; dash arrow
indicates gland; dotted arrow indicates collagen; and dash-dot-dash arrow
indicates epidermis. (Reprinted from the Ref. [236])
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In fact, a multimodal THz and optical imaging approach to the intraoperative

detection of non-melanoma skin cancers was proposed. Optical image acqui-
C

sition, performed in real time, would yield adequate resolution and contrast
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for analyzing skin morphology of all the regions highlighted in TPI; thus,
eliminating the false positive outcomes yielded by the THz pulsed imaging
alone, delivering practical solution to the intraoperative cancer detection.
CW THz imaging has also been successfully applied for non-melanoma
skin cancer delineation [173,237,238]. CW approach offers several advantages
over THz-TDS and THz pulsed imaging. In particular, CW systems are

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AN
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Figure 38: Images of a representative sample with BCC: (a) a THz power
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value image at 0.47 THz; (b) a H&E stained histology image of a 5-µm-
thick section of the tissue, where the tumor region is outlined with the black
solid line; (c) optical PLI data; (d) optical CPI data. Solid arrow indicates
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tumor site; dash arrow indicates gland; dotted arrow indicates collagen; and
dash-dot-dash arrow indicates epidermis. (Reprinted from the Ref. [236])
EP

cheaper, easier to operate and miniaturize. They are also easier to combine
with other imaging approaches. These factors are of major importance for
the clinical adoption. Several studies have shown that the frequency of ∼
C

0.5 THz yields the best contrast between cancerous and normal skin [236,242].
Therefore, it should be possible to improve the outcome of the THz method
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by optimizing the imaging frequency of the radiation.

The authors of the Ref. [173], were the first to propose THz polarization
imaging of the remitted THz radiation at 584 GHz for imaging cancer; exam-
ple of such images are presented in Fig. 40. Co-polarized and cross-polarized
THz images are shown in Figs. 40 (a) and (b), respectively. Cancer region is
indicated by a solid arrow in the THz cross-polarized image (Fig. 40 (b)) and

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AN
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Figure 39: A comparison of magnified images of the regions outlined with
solid squares in Fig. 38 (b): (a),(e),(i) 0.47 THz power value images;
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(b),(f),(j) a H&E stained histology images; (c),(g),(k) results of optical PLI;

(d),(h),(l) results of optical CPI. (Reprinted from the Ref. [236])
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with the black dotted line in the H&E histopathology image (Fig. 40 (c)).
The correlation between the co-polarized THz image (Fig. 40(a)) and the his-
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tology image is not satisfactory. The difference in the co- and cross-polarized
THz images is primarily due to the contribution of the specular reflection
of light to the co-polarized THz image. These reflections occur on the air
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cover glass and cover glass tissue interfaces due to the refractive index mis-
match. In contrast, cross-polarized THz imaging enables effective removal of
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the specular reflections, because the Fresnel component is co-polarized with

the incident radiation. It can be clearly appreciated that cross-polarized THz
image provides good contrast of cancer and reasonable signal to noise ratio.
As randomization of incident linearly polarized THz radiation, resulting in
the rise of the cross-polarized signal, is caused by multiple scattering of THz
radiation, it is clear that multiple scattering is not negligible in skin. Later

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Figure 40: Studying the tissue specimen with squamous cell carcinoma
(SCC): (a) a co-polarized THz reflectance image; (b) a cross-polarized THz
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reflectance image; (c) results of H&E stained histology of a 5-µm-thick frozen

section of the tissue; (d) cross-polarized optical image. (Reprinted from the
Ref. [173])
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work on colon tissue has extended the validity of these findings [250].
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Authors of the Ref. [173] were also the first to realize the role of com-
bining THz and optical imaging modalities and proposed a multimodal THz
and optical approach to delineating non-melanoma skin cancers. The optical
reflectance CPI from the Ref. [173] are presented in Fig. 40 (d). As com-
pared to the THz images (Figs. 40 (a) and (b)), the optical image of the
same specimen offers higher resolution. The comparison of the optical image

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(Fig. 40 (d)) to the histology image (Fig. 40 (c)) reveals a close correlation
of morphological features, as well as overall size and shape of the sample.
Moreover, the authors of Ref. [173] determined that the average reflectiv-

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ity values for non-melanoma skin cancers were lower as compared to normal
skin. In particular, the cross-polarized percentage reflectivity of the tumor
and normal regions, averaged over all the investigated samples, was found to

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be (0.69 ± 0.03)% and (0.84 ± 0.01)%, respectively. The difference in reflec-
tivity between normal and cancer was statistically significant (p < 0.001).
These results revealed that it could be feasible to find a common threshold

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value for cancer and normal skin.
The same research group have performed a clinical study evaluating the
combination of CW polarization THz and optical imaging of non-melanoma
skin cancers. Based on the results of the Ref. [173] it was possible to deter-

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mine the threshhold THz reflectivity values at 584 GHz for cancerous and
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normal skin and use those values for cancer delineation [237]. In addition,
they have proposed an algorithm for analyzing the multimodal images and
have proven that multimodal approach increases the sensitivity and speci-
ficity of the THz imaging alone. Statistical analysis confirmed the advantages
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of multimodal imaging as compared to the THz method alone. It has been
demonstrated that for terahertz imaging alone, the sensitivity and specificity
were 82% and 94%, respectively. For multimodal imaging, the sensitivity and
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specificity increased to 96% and 99%, respectively; indicating that terahertz-

optical imaging has great potential for the intraoperative assessment of tumor
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margins.
In Refs. [257, 258], the use of THz for diagnosis of breast tumors has
been considered. Significant contrast has been observed in THz dielectric
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properties and images of three tissue types – tumor, fibrous and fat tissues.
Fig. 41, demonstrates the results of THz pulsed imaging of freshly excised
breast tumor in vitro reprinted from the Ref. [258]: (a),(b) represent the THz
parametric images calculated via two different approaches of THz waveform
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processing; (c) shows the results of histological examination of tissues. The

origin of the observed contrast in the THz images is primary associated with
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enhanced water content in abnormal tissues. The THz-TDS and THz pulsed
imaging could be applied for intraoperative mapping of breast tumors to
maximally preserve normal tissues from resection, and, as a consequence, to
reduce the cosmetic harm caused by surgery of breast tumor.
Remarkable progress has been reported in the THz intraoperative diag-
nosis of malignant brain tumors [247, 259–261]. The THz reflection-mode

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images of freshly-excised and paraffin-embedded tissues of the whole brain of

the rat model have presented significant contrast between normal and malig-
nant tissues and a good correlation with magnetic resonance images (MRI)

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of the brain [247]. This contrast has been reported to originate from high
water content and abnormal cell density in malignant tissues [247, 260]. An
in-depth analysis of the THz reflectivity and dielectric response of paraffin-

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embedded normal brain tissues and glioma models from mice reveals optimal
THz frequencies, at which these classes of tissues could be differentiated us-
ing various THz imaging modalities, including the intensity and coherent CW

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THz imaging, and the THz pulse imaging [259]. In particular, for paraffin-
embedded samples, the real part of the dielectric permittivity provides a
contrast between normal and pathological tissues up to 15% at the frequen-
cies above 1.5 THz, while the imaginary part yields a contrast, which is

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higher than 20%, in the spectral range below 1.0 THz.
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The intraoperative diagnosis of malignant brain gliomas using the THz
reflection-mode imaging has been systematically analyzed in Ref. [261]. This
study involves measurements of glioma models from mice in vitro and in
vivo and human brain gliomas in vitro; while gliomas of different grades have
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been considered. In Fig. 42, we compare results of the visualization of the
glioma model from the mice using different imaging modalities, one of which
is the THz reflection-mode imaging (TRI). The observed results highlight
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the potential of the THz technology in label-free and real-time intraoperative

assessment of gross total resection of low- and high-grade brain gliomas. This
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Figure 41: Results of the THz pulse imaging of freshly excised breast tu-
mor in vitro: (a),(b) THz parametric images calculated via two different
approaches of THz waveforms processing; (c) results of histological examina-
tion of tissues. (Reprinted from the Ref. [258])

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could become a significant advantage of THz biomedicine, since, nowadays,

it is difficult to discriminate clearly a tumor borders during neurosurgery
using conventional instruments of intraoperative diagnosis, such as MRI or

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fluorescence spectroscopy and imaging [262].

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Figure 42: The results of discrimination of healthy brain tissues and glioma
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model from 4 mouse brain samples using various imaging modalities: (a) pre-
operative MRI images in living mouse; (b) White Light images (WLI) of the
excised brain; (c) fluorescence images using green-fluorescent protein (GFP);
(d) results of H&E stained histology; (e) Optical Coherence Tomography
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images (OCT); (f) THz reflection-mode images (TRI); (g) 5-ALA-induced

ppIX fluorescence images. The TRI images allow for detection of the tumor
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margins. (Reprinted from the Ref. [261])

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6.5 Terahertz technology to aid histology

Along with the discussed approaches for noninvasive, least-invasive and

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intraoperative THz diagnosis of malignancies, which predominantly involves
measurements of tissues in vivo, THz spectroscopy and imaging also has po-
tential for accompanying histological examination of excised tissues to detect
the margins of malignancy [263]. THz measurements of histological samples

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could be performed as during the intraoperative express histology (for ex-
ample, during surgery of malignancies of the skin [238, 240, 241, 250], the
breast [257, 258], and the brain [247, 259–261]), as after the surgery in order

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to roughly distinguish various classes of tissues in an automatic regime for
minimizing the amount of tissues needed to be examined manually via the
microscopy.

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Besides the technical problems associated with the development of spe-
cialized THz instruments and related signal processing approaches, the THz
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accompanying of histological examination of tissues needs the development
of novel techniques for tissue fixation. Recently, numerous approaches to fix
the tissues for improved THz measurements have been considered; among
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them are

• standard formalin fixation [264];

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• gelatin fixation, which preserve tissue from hydration/dehydration and

sustain its THz dielectric response close to that of tissues in vivo [265];
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• dehydration of tissues involving the use of various agents in order to

increase the depth of measurements [266] and reveal non-water-related
differences of normal and pathological tissues [267];
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• fixation in paraffin emulsion [263] and paraffin embedding [259], which

reveal structural differences of normal and pathological tissues;
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• freezing the tissues (see section 5.7) [268].

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In Fig. 43, we demonstrate microscopic images and reflection-mode THz

pulsed images of 10-µm-thick sections of tissues, reprinted from the Ref. [263].
The observed results justify an important of optimal tissue fixation for the
THz measurements; here, an improved contrast between healthy tissues and
cancer as well as a better correlation with the data of visual microscopy are
observed for the tissue sections fixed in a paraffin emulsion.

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6.6 Double-Debye model of terahertz dielectric response

of tissues

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It is notable that the applying of expression for water permittivity to
description of biological tissue permittivity turns out surprisingly productive.
A convenient approach for describing the dielectric response of tissues in

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vitro and in vivo is associated with the use of the double-Debye model of
complex dielectric permittivity [233] comprised of the two Debye relaxation
terms (see Eq. (8)), where ε∞ , τ1 , τ2 , ∆ε1 and ∆ε2 have the same physical

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meaning but another numerical values. Table 2 depicts the double-Debye
model parameters for tissues in vitro and in vivo from a number of Refs.,
while the model parameters for water are shown as a reference to compare
with. From this Table one could see that the double-Debye models have been

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constructed for healthy and malignant tissues of the skin and the breast, while
estimating the model parameters for healthy and abnormal tissues from other
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localization has not been carried out yet – it is still of high importance.
We observe low-to-moderate differences between the THz dielectric per-
mittivity models of water and tissues, which justifies significant contribution
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Figure 43: A comparison of (a),(b) the visible microscopic images and (c)-(h)
the reflection-mode THz pulsed images of the H&E-stained histopathologic
sections, where the pairs of the THz images (c) and (d), (e) and (f), (g) and
(h) are obtained after fixation of the tissues in paraffin, water, and paraffin
emulsion, respectively. (Reprinted from the Ref. [263])

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of high water content to the THz dielectric response of tissues. The other
components of tissues, as well as bonding and segregation of water might be
origins of differences in picosecond dynamics of water and tisues. The no-

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ticeable spread in relaxation times in Tab. 2 might occur from the difference
in the measurement techniques and data processing approaches, as well as
from the different experimental conditions, such as temperature, humidity,

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method of tissue fixation, which was employed in studies in vitro, and even
occlusion effects, which is inherent for measurements in vivo [275].
The double-Debye model yields parametrisation of the results of THz

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Table 2: Parameters of the double-Debye models of the THz dielectric re-
sponse of tissues; here, BCC stands for the basal cell carcinoma of the skin,

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BFT and BTT stand for fibrous tissue and tumor of the breast, respectively.
(Courtesy of M.M. Nazarov)

# Object ε∞
AN ∆ε1 ∆ε2 τ1 , ps τ2 , ps Ref.
1 Water 3.30 75.00 1.90 8.50 0.17 [269]
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2 Water 3.50 73.50 1.40 8.20 0.18 [16]
3 Water 4.10 72.20 2.50 10.60 0.18 [233]
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4 Water 3.20 73.60 1.60 8.00 0.18 [25]

5 Water 3.42 74.16 1.38 7.87 0.18 [270]
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6 Skin in vitro 2.89 20.34 1.74 3.82 0.11 [271]

7 Skin in vitro 2.58 10.54 1.58 1.45 0.06 [272]
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8 Skin in vitro 2.86 25.70 1.76 4.80 0.10 [273]

9 Skin in vivo 3.00 56.40 0.60 10.00 0.20 [233]
10 Skin in vivo 3.00 54.40 0.60 9.40 0.18 [274]
C

11 BCC in vitro 2.90 28.53 1.90 4.35 0.11 [271]

12 BCC in vitro 2.58 13.37 1.58 1.55 0.06 [272]
AC

13 BCC in vitro 3.02 72.25 1.95 11.03 0.13 [273]

14 BFT in vitro 2.10 72.60 1.80 10.30 0.07 [25]
15 BTT in vitro 2.50 73.60 2.80 9.10 0.08 [25]

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spectroscopy of tissues using only 5 coefficients - ε∞ , ∆ε1 , ∆ε2 , τ1 , and τ2 .

This capacious physical representation of the complex dielectric permittivity
allows the understanding of picosecond relaxation dynamics in tissues. It

PT
could be used in order to simulate the THz wave – tissues interactions using
analytical and numerical methods of computational electrodynamics [25,233,
274], and to model the THz spectroscopy and imaging of tissue employing

RI
different THz instruments [276]. Obviously, the spectral range of this model
applicability for describing the THz wave – tissues interactions are limited
with rather low frequencies of about ν ≤ 0.25...0.33 THz. (depending on

SC
the type of tissue), at which the effects of THz wave scattering on tissue
inhomogenities are negligible; while, for higher frequencies, the structural
inhomogenities of tissues and the Mie scattering effect should be taken into
account.

U
Finally, another remarkable advantage of experimental data parametri-
AN
sation with the double-Debye model is associated with an ability to use the
coefficients ε∞ , ∆ε1 , ∆ε2 , τ1 , and τ2 as physical principal components for
differentiation between healthy tissues and malignancies [271].
M
6.7 Challenges in THz diagnosis of malignancies
In this section, we have discussed numerous applications of the THz spec-
D

troscopy and imaging in medical diagnosis of malignancies, including non-

invasive, least-invasive and intraoperative diagnosis, as well as accompani-
TE

ment of histology. Despite the considerable progress in this area of THz

biophotonics, numerous problems still restrain the transfer of the THz di-
agnosis techniques to medical institutions. Along with rareness, complexity,
cumbersomeness and high cost of the THz instruments, we notice the follow-
EP

ing:

• The development of novel methods and instruments for the THz diag-
C

nosis of malignancies requires systematic measurements and statistical

analysis of the THz responses of normal and pathological tissues, ei-
AC

ther in vivo or ex vivo. This analysis would help to both select the
optimal spectral ranges and principal components for the differentia-
tion between healthy and malignant tissues, and analyze sensitivity,
specificity, type I and II errors. At the same time, most of the existing
research dedicated to the THz diagnosis considers only small numbers

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of tissue samples, which yields only preliminary results (feasibility test)

before committing to the required full-blown study.

PT
• Further developments in the area of THz diagnosis (especially, the least-
invasive and intraoperative diagnosis of the internal organs) is consid-
erably complicated by the absence of the THz waveguides and fibers,

RI
which allow for delivering the THz waves to/from the hard to access
tissues (see Sec. 4.4). Developing the THz waveguides and fibers, along
with adapting the existing developments for the needs of the THz di-

SC
agnosis, would become a significant contribution to THz biomedicine.

• The spatial resolution of modern THz spectroscopic and imaging sys-

tems is diffraction-limited, being the function of electromagnetic wave-

U
length λ and numerical aperture NA of the optical system (see Sec. 4.3).
The limited spatial resolution decreases an accuracy of the tumor mar-
AN
gins’ detection. Thus, the development of novel methods for the high-
resolution THz spectroscopy and imaging is of a significant importance
for the THz medical diagnosis.
M
• Finally, a prospective approach for improving the efficiency of non-
invasive, least-invasive and intraoperative diagnosis of malignancies is
associated with combining the information from several instruments
D

for the abnormal tissue detection. For example, the methods of THz
spectroscopy and imaging could be combined with the modern tools
TE

of tissue visualization in visible and near-infrared spectral ranges, such

as OCT, fluorescence spectroscopy and imaging, polarization-sensitive
imaging, opto-acoustic sensing, Raman spectroscopy and imaging, etc.
EP

Nevertheless, a unique ability for the label-free, fast and accurate discrim-
ination of healthy and malignant tissues in a number of localizations does
not make a doubt that all the listed challenging problems would be miti-
C

gated with a rapid progress in the THz science and technology; leading to
THz spectroscopy and imaging being applied to the diagnosis of malignancies
AC

in clinical practice.

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7 In vivo terahertz sensing of eye cornea

7.1 Introduction

PT
Eye diseases are one of the most important health problems in the world.
According to the World Health Organization, more than 300 million people in
the world suffer from different eye diseases. Dry eye syndrome or corneal and

RI
conjunctival xerosis is a complex disease that is prevalent all over the world
and becomes one of the major problem of modern ophthalmologic pathology.

SC
According to Russian researchers, up to 12% of ophthalmic patients under
40 years of age and over 67% of patients older than 50 years suffer from this
disease [277]. The concept of ”dry eye syndrome” is defined as a complex of
signs of the corneal and conjunctival epithelium lesions due to a decrease in

U
the quality and/or amount of tear fluid [278]. This fluid forms a tear film on
the eye surface, which performs a number of important functions, including
AN
trophic, protective and optical. Thus, a violation of the composition or
production of the tear film can lead to sufficiently serious damage to the
anterior eye surface.
M
The diagram representing the anterior eye fragment construction is shown
in Fig. 44. From front to back the anterior eye section consists of 6 to 12-µm-
thick three-layer pre-corneal tear film and 450 to 650-µm-thick (central part)
D

five-layer cornea. The cornea consists of the epithelium, the Bowman’s mem-
brane, the stroma, the Descemet’s membrane and the endothelium [279]. The
TE

major corneal component – stroma takes 90% of its total thickness. The typi-
cal water content in stroma is 75–80% that exceeds the water content level in
any other types of connective tissue besides cartilage that has a similar water
content [280]. This is determined by the presence of negative charges on the
EP

chains of glycosaminoglycans, which attract a positively charged dipole of

the water molecule. Impaired hydration of glycosaminoglycans chains dete-
riorates their size, deforms the distance between collagen fibers and, thus,
C

breaks the well-ordered structure, which leads to the loss of cornea trans-
parency. Metabolic processes in the cornea should maintain the transparency
AC

of corneal tissue and prevent its violation [281]. The pre-corneal tear film
transports metabolic products to and from the cornea, prevents the cornea
from drying, lubricates the ocular surface and has anti-bacterial properties.
The middle aqueous-layer of the tear film is represented by water solution
and makes up 90% of the tear film volume. Under the normal conditions,
the tear fluid of the ocular surface and in the aqueous humor of the anterior

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PT
RI
U SC
AN
M
D

Figure 44: Diagram of the anterior eye fragment construction. (Courtesy of

I.A. Ozheredov)
TE

chamber is isotonic with the corneal stroma. However, the hygroscopicity

of glycosaminoglycans regulates the flux of water from the tear film through
EP

the corneal epithelium (passive transport) into the corneal stroma.

The basis of dry eye syndrome progression is a dysfunction of the pre-
corneal tear film. Its assessment is a leading direction in the diagnosis of
C

this disease. In clinical practice, the most common method for determining
the stability of the tear film is Norn test [282]. For this test, 0.1% solution
AC

of fluorescein sodium salt is typically used. The study is conducted at the

slit lamp with a cobalt-blue filter. The time of appearance the gaps into
the stained tears is recorded. The conjunctival disease, which develops as a
result of hypolacrimia, usually manifests itself by tearing of the tear film in
the corneal epitheliopathy [283].
Non-invasive methods for studying the stability of a tear film include

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thioscopy [284]. The method allows to evaluate several parameters: tear

film stability, thickness of the lipid layer of the tear film, quality of the
original layer when wearing contact lenses. Confocal microscopy expands

PT
the possibilities of studying the anatomy of the cornea at the level of its
microstructure [285]. The method allows real-time studying of each corneal
layer and detecting the changes of the corneal structure. This is especially

RI
important in the subclinical course of the dry eye syndrome, when traditional
methods of research are poorly informative. It has been established that with
the dry eye syndrome the main changes affect the epithelial layer of the cornea

SC
in the form of edema and cell polymorphism. With the existing merits of the
method, its drawback is the contactness of the study and the likelihood of
negative effects of the analgesic on the epithelium of the eye surface during
the study.

U
The first in vivo application of THz spectroscopy and imaging for the
AN
corneal tissue hydration sensing was recently reported in Ref. [286]. Authors
used THz pulsed imaging and millimeter-wave reflectometry system to ac-
quire data for five rabbit corneas. The paper reported a good correlation
between the corneal thickness and reflectance coefficients. For slight dehy-
M
dration of healthy cornea, a gentle stream of air and a Mylar window was
employed. However, the method used in the work described above, is still
far from being a practical device. THz optical design proposed in Ref. [287]
D

yields acquisition of the THz reflectivity maps of in vivo cornea without any
flattening window. The authors suggested beam optics design for scanning of
TE

a curved eye cornea surface at normal incidence, while keeping the source de-
tector and target stationary. Such THz system might be used for non-contact
THz imaging of animal and human eye.
EP

Thus, we discuss the application results of THz reflectometry technique

for measuring the dielectric permittivity of corneal sample for different hydra-
tion levels. We propose a procedure based on normalization of the measured
value of cornea sample permittivity on the parameters of highly porous trans-
C

parent material – nanostructured aluminum oxyhydroxide as a tissue phan-

tom. Frequency-domain THz reflectometry system will be used for in vivo
AC

sensing of human dynamic of precorneal tear film thinning.

7.2 Determination of cornea dielectric permittivity

The dielectric constant of the corneal tissue is determined by mainly three
of its components: collagen fibers, bound water, and free water. To evaluate

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the effective dielectric constant of hydrated corneal medium, the effective

medium approach, in which it is related to the dielectric constants of dry
cornea and water, was suggested in Ref. [288]. Such approach, based on the

PT
Bruggeman model (see section 2.3), considers a medium composed of two
equivalent components – the unbound water and the collagen fibers. The
dielectric permittivity of the entire medium εeff meets the Eq. (25).

RI
To simplify modeling we propose to use highly porous transparent ma-
terial - nanostructured aluminum oxyhydroxide (NAO) - as a tissue phan-
tom for calibration of measurements. The dielectric properties of NAO was

SC
studied in details in Ref. [289], accounting for water chemically bound and
adsorbed by NAO fibrils. The NAO was pressed into pellets and, then,
used as a reference for calculation of the dielectric response of the hydrated
corneal tissue. To minimize the impact of spatial dispersion on the results,

U
the pellet’s shape emulated the human eye shape.
AN
For the experimental determination of the dielectric constant of corneal
tissue, we used the reflection-mode THz-TDS setup, described in details in
Ref. [289]. It allows for measuring the instantaneous electric field amplitude
of a sub-picosecond-duration THz pulse. The shape of the temporal profile
M
of the alternating electric field gained information about both the amplitude
and the phase of the THz radiation in a broad spectral range. Another
phantom of human eye was used as a sample. It was based on the human
D

cornea sample – cadaver corneal disc. The corneal disk was mounted on a
plastic hermetically sealed hollow chamber with a spherical surface and a 8-
TE

mm-diameter aperture. The design of the holder allowed one to fill it with a
0.9% solution of NaCl to create pressure on the sample from the inside. Thus,
the system ”sample-holder” provided wetting endothelium of corneal sample.
EP

Due to pressure created by liquid in the holder, the sphericity of the corneal
sample is maintained during the experiment. The pipette was used to drop
water onto the anterior surface of the cornea, to keep it moisturized, imitating
the blinking effect. The phantom eye was installed onto the balance providing
C

the sensitivity better than 0.1 mg. The balance allowed for controlling the
phantom weight and for monitoring its change due to the water evaporation
AC

from the cornea. Such change of the weight might be recalculated into the
cornea dehydration level. The THz beam from the experimental setup was
focused into the cornea under study by a wide aperture lens with the focal
distance of 50 mm. The diameter of the THz spot at the sample surface was
about 4 mm. The reflected THz signal was collimated by the same lens and
collected by detector.

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By using the proposed phantom eye on the sample place, we obtained

a series of the THz reflection spectra from the cornea with simultaneous
control of the sample weight. The reduction of the sample weight and the

PT
water content in corneal tissue leads to the reduction of the THz spectral
amplitude. In case of dehydration of the corneal tissue within the small
range the unbound water content decrease, while the bound water remains

RI
the same [290]. Thus, the changes of the THz reflectivity due to cornea
dehydration are primarily associated with the decrease of unbound water
content in a corneal matrix.

SC
The measured THz spectra for each hydration level were normalized by
the spectra obtained for the NAO pellet. This normalization process can be
described by the following Eqs.:

U
Ac
Aw = , ϕw = ϕc − ϕNOA , (52)
ANOA
AN
where Aw , Ac , ANAO and ϕw , ϕc , ϕNOA are spectral amplitudes and phases
of water in the corneal matrix, the entire cornea and the NAO media, re-
spectively. Such a normalization allows us for excluding the contribution of
M
bound water and corneal collagen matrix to the dielectric permittivity of a
corneal sample. Finally, the dielectric response is calculated as follows
1 + zw
D

εw = , (53)
1 − zw
TE

where
Aw Aw + tan ϕw
zw = q + iq . (54)
1 + tan2 ϕw 1 + tan2 ϕw
EP

Real and imaginary parts of the complex dielectric permittivity of a corneal

sample for different hydration levels obtained via the described procedure is
shown in Fig. 45.
In Fig. 45, the curve corresponding to the highest hydration level of 81%
C

was measured immediately after taking the phantom eye out of the corneal
storage solution. The next scan related to 80% was taken in 12 min after
AC

the first one. The remaining two graphs corresponding to 75% and 73% of
cornea hydration were obtained in 82 and 100 min, correspondingly. A clearly
detectable difference in the measured curves highlights a high sensitivity of
the proposed technique, especially in the low frequency region.
It should be mentioned that despite all the data were obtained for a sam-
ple, whose hydration varied within the physiological norm [281], the change

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in phantom hydration level occurred in a long time (more than an hour).

During this time, a change in the hydration of a corneal tissue might be
associated with an additional change of the sample geometry. In order to

PT
correctly separate the mechanisms that lead to a change in the reflected THz
signal, additional control measurements are necessary, for example, measure-
ments of the cornea thickness, as was suggested in Ref. [286].

RI
7.3 In vivo real-time applications

SC
The described THz-TDS-based reflectometry is proven to be a good tool
for evaluation of the dielectric parameters of corneal tissue. But due to the
system complexity and the delay-line limitations, this technique is not reliable
for the in vivo real-time applications. Moreover, during measurements, the

U
thickness, composition and other parameters of the tear film are significantly
altered. Thus, it is necessary to select a technique for the rapid measurement
AN
of cornea and tear film reflectivity in the THz range.
A possible solution is associated with the use of the THz emitter / coher-
ent receiver concept [292], allowing to build a compact and low-cost CW THz
M
reflectometer. On the source side two fiber-based distributed feedback CW
diode lasers were precisely temperature controlled for generation of a narrow
(10 kHz) line in the range of 1530–1608 nm with the average power of 22 mW
D

each. The laser light was combined by the X-type fiber optical beam splitter
TE

8 8

81 % 81 %
EP

6 80 % 6 80 %

75 %

73 %
Re( )

4 4
Im(
C

2 2
AC

0 0
0,2 0,4 0,6 0,8 1,0 0,2 0,4 0,6 0,8 1,0
f, THz f, THz

Figure 45: Real (a) and imaginary (b) parts of the complex dielectric per-
mittivity of a corneal sample for different hydration levels. (Reprinted from
the Ref. [291])

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with the splitting ratio of 50/50. The outputs of the splitter were connected
to the THz emitter and receiver, respectively. We used an ”off-the-shelf”
low-temperature InGaAs bow-tie photoconductive antenna. The pulsed DC

PT
bias voltage for the THz emitter with the amplitude of 6 V and the frequency
of ∼ 40 kHz was supplied by the function generator. The emitted THz signal
was, first, guided through the polyethylene lens to the sample and, then,

RI
after the reflection, it was transmitted through the second polyethylene lens
to the receiver. The resulting photocurrent was measured by a digital lock-
in amplifier (SR-830, Stanford Research Systems). The difference frequency

SC
range of lasers could be be tuned in a range of 50–300 GHz. In this range, the
described photoconductive-antenna-based frequency-domain system delivers
only a sub-microwatt power, but that is enough for our study.
During the in vivo experiments, the human’s head was fixed by the special

U
ophthalmological stage [291]. The radiation at the frequency of 100 GHz
AN
was focused onto the corneal surface by the lens with the focal distance of
30 mm. The ophthalmological stage ensured the matching of the examined
cornea and the focal plane of the THz lens. The specularly reflected signal
was collected by the lens with a similar focal distance and detected by the
M
second photoconductive antenna. Test trials of the proposed method were
performed on the eyes of several males, aged 22 to 45 years, whether they
need to wear glasses or not. For each human eye, we measured the reflected
D

signal versus time. Each measurement started at the time of eye opening.
During the measurement series, the subjects were requested to keep the eye
TE

open as long as possible, while they were able to close the eye as soon as they
needed to. The measurements continued until the next eye closing.
The typical dynamics of the observed eye reflectivity is shown in Fig. 46.
EP

There are two well recognized areas corresponding to the open and closed
eye. The THz reflectivity of the open eye demonstrates the dehydration
dynamics. Such dependency decreases with time and can be fitted by a linear
function. Each time the eye was closed the reflectivity decreases significantly
C

and remains constant while the eye is closed. Then, after eye opening, the
reflectivity restores to the value similar to that observed in the previous cycle.
AC

Thus, in the Fig. 46, we observe the physiological thinning of the pre-corneal
tear film between blinks, followed by the restoration of thickness of the tear
film to the its initial value. The results of the described preliminary studies
justify an ability for sensing the pre-corneal tear film dynamics using the
proposed CW THz reflectometer.
The abovementioned test measurements showed the sensitivity to the var-

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ious physiological dynamics of the tear film thinning. The dysfunction of the
pre-corneal tear film is mainly related to the dry eye syndrome progression.
In vivo measurements of changes in the THz reflectivity due to the pre-

PT
corneal film thickness variation allows for controlling such progression. The
physiological connection between the cornea and the tear film is due to the
peculiarities of the formation of the tear film itself, in which corneal epithe-

RI
lial cells take an active part. On the other hand, the formation of glycocalyx
due to microvilli of epithelial cells and transmembrane mucins allows one to
keep a tear film on the ocular surface and hinder the development of xerotic

SC
changes. Thus, the problem of studying the change in the hydration of the
cornea is impossible without taking into account the state of the pre-corneal
tear layer. For the real evaluation and estimation of the criteria for dysfunc-
tion determination, further full-blown study should be accompanied with the

U
approved clinical measurements.
AN
Thereby, in this section, we have demonstrated that the measured THz
dielectric response (and, thus, THz reflectivity) provides an important in-
formation about the physiological state of the corneal tissue and the human
eye. We have shown that the THz reflectometry is capable for in vivo sens-
M
ing of the tear film thinning kinetics. The CW THz reflectometry was sug-
gested as a promising real-time measurement technique reliable for clinical
applications; this method differs significantly from the existing and currently
D

R
TE

0,12 0,12
EP

0,08 0,08

0,04 0,04
C

0 10 20 30 40 50 0 5 10 15 20
AC

Figure 46: In vivo measurements of the THz reflectivity of an eye cornea:

(left panel) 45-year-old man not wearing glasses; (right panel) 26-year-old
man wearing glasses. Here, points stand for the experimental data, while
lines represents the linear approximation. (Reprinted from the Ref. [291])

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available methods of ophthalmological diagnosis.

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8 Color vision and terahertz radiation
8.1 Introduction

RI
Methods of THz imaging are interesting for a number of applications
related to the detection and localization of particular substances in an object
under study. The fact that many organic substances have strong absorption

SC
bands in the THz range allows for detecting and identifying these substances
using the THz imaging. In visible and IR domains, the imaging data is
often represented by pseudo-color images, in which low pixel brightness is in

U
blue, while high brightness is in red, or vice versa. Generally, this approach
offers an opportunity to improve the image perception; however, it has a
AN
number of limitations, especially, when one needs to identify a substance.
In his case, the result depends strongly on the illumination conditions, and
the spectral contrast between several substances in a sample; thus, impeding
identification of these substances with similar or overlapped spectra.
M
One way to improve the information content of an image is to increase the
number of channels, in which images are acquired, by the spectral selection of
images through illumination or in the detection process. Multispectral laser
D

imaging combines a narrow-band illumination, strictly corresponding to the

spectral features of an object, and standard image acquisition methods. It
TE

can be used to identify substances of interest using their spectral signatures.

For example, Brydegaard et al. [293] described a system, in which an ob-
ject could be illuminated using 13 independent light sources. In this system,
EP

spectral discrimination is ensured by sequentially turning on each source.

A combination of images from various spectral ranges makes it possible to
significantly improve the clarity of images acquired using a multichannel sys-
C

tem. Practical application of a multichannel image acquisition was described

by Clancy et al. [294], who utilized it for studying a large intestine dur-
AC

ing surgery: multiple images were used to monitor the relative hemoglobin
concentration in a tissue. In addition, they demonstrated an ability for the
intraoperative monitoring of the arterial blood content in tissues.
Watanabe et al. [295] proposed to take into account the spectral infor-
mation in the acquiring THz images for nondestructive chemical analysis of
an object. They substantiated a method for analysis of multispectral THz

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images, in which each point contained the transmission spectrum of the ob-
ject. The method was implemented in the spectral range of 1.0–2.0 THz
and involved the use of known THz absorption spectra of the substances to

PT
be detected. The method was developed further by Shen et al. [296], who
obtained THz images using broadband (0.06–4.00 THz) THz pulses reflected
from the object of interest. If the characteristics of an object are unknown,

RI
it is necessary first to reveal inhomogeneities in its image and then, using
image phase analysis results, to turn to a targeted examination of the region
of interest. For example, to acquire a THz image, Shen et al. [296] used

SC
broadband illumination possessing up to 60 independent spectral channels in
the bandwidth of ∼ 3.0 THz with the spectral resolution of 50 GHz. This
makes possible detection of various substances, while that knowledge of the
absorption spectra of substances in an object was unnecessary.

U
In this section, we discuss further development of THz imaging technol-
AN
ogy, based on the concepts of multispectral and multi-temporal domains,
which combine the principles of color vision, phase analysis and tomography
in THz image acquisition. We discuss application of these modalities of THz
imaging.
M

8.2 Experimental apparatus and samples

D

To obtain THz images, we used a commercial THz-TDS – namely, the

TPS Spectra 3000 from TeraView Ltd, equipped with a module for the reflec-
TE

tion imaging. This configuration yields detection of the THz pulses featuring
a broad spectrum in range of 0.06 to 4.00 THz, after their reflection from an
object. During imaging, the object is placed in a horizontal plane, onto a
motorized X–Y translation stage. The translation stage allows for scanning
EP

the surface of an object with a focused THz beam in the area of 80 × 80 mm.
The imaging results have the form of a 3D array E (xi , yj , tk ), each pixel of
which is proportional to the amplitude of electric field of the THz waveform,
C

came from a (xi , yj )-point of the sample surface with a tk -delay. Standard
software supplied with the spectrometer allows one not only to analyze mea-
AC

sured 3D arrays/images for each individual point (xi , yj ) by examining the

temporal profile E (t) of the reflected signal or its Fourier spectrum E (ν),
but also to map out sections through an image: a time-domain section at
a particular instant in time; or a spectral section at a particular frequency.
A point-by-point analysis of the spectrum of the reflected signal makes it
possible to determine the chemical composition at each sample point; while

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an analysis of the temporal profile provides information about the internal

structure of the sample. If a spectral feature characteristic of the substance of
interest is found in the spectrum of the reflected signal, a spectral section at

PT
some characteristic frequency allows one to visualize the spatial distribution
of the substance over the entire image.
In addition to a point-by-point analysis of a sample, one can map out a

RI
time-domain or frequency-domain section through its image – i.e., to carry
out layer-by-layer analysis in the frequency and time domains. It is worth
pointing out that in the case of a point-by-point analysis of an image, spec-

SC
tral analysis cannot be carried out simultaneously over the entire image. A
layer-by-layer analysis of spectral sections through an image at a particular
frequency, in turn, offers the possibility to visualize the presence and spa-
tial distribution of a substance that has a spectral feature at that frequency,

U
which makes it impossible to simultaneously detect and visualize several dis-
AN
tinct substances. Also, analysis of a time-domain section is incapable of
simultaneously visualizing inhomogeneities located at different depths with
respect to the sample surface. To overcome these limitations, we proposed
and tested the concepts of multispectral and multi-temporal domains in THz
M
imaging. The use of these concepts allowed us to improve image contrast
and considerably reduce the time needed to detect and localize substances of
interest and hidden objects. To test the proposed concept, we prepared and
D

studied the following samples:

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• arabinose, lactose and hydroperit powders packed in polyethylene bags;

• a sandwich structure comprised of three metallic discs located between

cardboard layers;
EP

• a skin sample ex vivo with BCC.

C

8.3 Multiband terahertz imaging in spectral domain

The concept of multispectral-domain in THz imaging is based on a con-
AC

tinuous layer-by-layer summation of spectral sections through an image and

a superposition of specially designed digital spectral filters, whose position
is determined by the spectral features of a sample. In Fig. 47, we illus-
trate the concept of the multi-spectral THz imaging using the data of THz
reflection-mode measurements of arabinose, lactose and hydroperit powders
packed in polyethylene bags. These substances cannot be distinguished in

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the photographs of the bags, shown in Fig. 48. The THz spectra of the signal
reflected from the powders and the positions of three digital filters (red – R;
green – G; blue – B) with the Gaussian profiles are shown in Fig. 47. For

PT
each filter, the following integral is calculated point-by-point
Z
(j)
Yi = Ei (ν) Fj (ν) dν (55)

RI
ν

here, Ei (ν) is the spectrum of the THz pulse at the ith point of the image;
Fj (ν) is the j th digital filter; j = 1, 2, 3 in case of using three channels; and

SC
three numbers Ri , Gi and Bi are assigned to each pixel of the image, corre-
sponding to the red, green and blue colors. An example of multi-spectral THz
image is presented in Fig. 49, where the three substances are easy to distin-

U
guish. It is worth pointing out that the presence of interference oscillations
in the spectra in Fig. 47 and the nonuniform coloration of the homogeneous
AN
substance in Fig. 49 are caused by the influence of the packaging and the
nonuniform thickness of layers of substance in the bags.
Note that, in the case of 24-bit color images, the numbers Ri , Gi , and Bi
should be normalized. We considered the following schemes for performing
M
the normalization procedure:

1. At each ith point of the image, we find the maximum and minimum
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color components – max [Ri , Gi , Bi ] and min [Ri , Gi , Bi ]. The normal-

(j)
ized value of the j th color component at the ith point Yi is calculated
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C EP
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Figure 47: THz reflection spectra of (1) arabinose, (2) lactose and (3) hy-
droperit powders in polyethylene bags, overlapped with digital spectral filters
featuring the Gaussian profiles. (Reprinted from the Ref [289])

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Figure 48: Photographs of the bags containing arabinose, lactose and hy-
AN
droperit powders. (Reprinted from the Ref. [289])

(j)
from its unnormalized value Yi as follows:
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(j)
(j) Yi − min [Ri , Gi , Bi ]
Yi = × 256. (56)
max [Ri , Gi , Bi ] − min [Ri , Gi , Bi ]
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Figure 49: A multi-spectral THz image of the bags containing arabinose,

lactose and hydroperit powders. (Reprinted from the Ref. [289])

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Figure 50: Schematic of a sandwich structure comprised of three metal discs

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located between cardboard layers. (Reprinted from the Ref. [289]

This point-by-point normalization scheme allows one to extend the dy-

SC
namic range of the color image, enhance the color depth, increase the
number of color hues, and visually distinguish more details in image.

2. As distinct from the above scheme, global normalization is performed –

U
i.e., the global extrema over the image max [R, G, B] and min [R, G, B],
AN
are used for normalization, instead of the point-by-point maxima and
minima of the color components. This normalization scheme allows one
to more effectively solve the problem of phase analysis when seeking
uniform inclusions against an unknown background, where the visual-
M
ization of their specific details in images is not very important.

Thus, an appropriate scheme is chosen for performing the normalization

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procedure, depending on the imaging purpose, and each pixel of the im-
age is assigned a particular color, depending on the spectral features of the
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substance at this point.

To effectively detect and localize substances of interest, it is reasonable
to place filters in spectral regions with the most prominent features, because
this leads to an increase in contrast and ensures a difference in color and
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hue between the substances to be detected. An optimal choice of the set of

digital filters allows one to visualize the presence of spectroscopically different
substances in a sample.
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8.4 Multiband terahertz imaging in temporal domain

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The concept of multi-temporal domain in THz imaging is an analogue of

the concept of multi-spectral one; it is based on a continuous layer-by-layer
summation of time-domain sections through an image and a superposition
of specially designed digital time-domain filters.

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The multi-temporal THz imaging modality was used to analyze the tem-
poral profile of a THz signal reflected from a layered object, shown in Fig. 50.
In Fig. 51, we demonstrate the temporal profile (THz waveform) measured

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at a single point of such a structure; it contains a set of pulses representing
reflections from different cardboard layers. Furthermore, in Fig. 51, we show
digital filters that were used for multi-temporal THz imaging. Finally, in

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Fig. 52, we present the multi-temporal THz image of the layered object, on
which we could clearly observed all its sub-surface component. Their colors
are straightforwardly determined by their depths in the sandwich.

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8.5 Multiband terahertz imaging in the spectral and
temporal domains for early skin cancer diagnosis

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To conclude, we present multispectral and multi-temporal THz imaging
AN
results for studies of biological tissue, illustrated by the example of the diag-
nosis of a human skin sample for skin cancer detection [289]; see Fig. 53. To
detect skin lesion and localize the lesion focus, we used histological exami-
nation results; Fig. 53 (a). In Fig. 53 (a), the skin area affected by cancer
M
is clearly seen. In Figs. 53 (b) and (c), we show the multi-temporal and
multi-spectral THz images of the skin sample.
It is worth noting that the ability to detect BCC using reflection spectra
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of broadband THz pulses depends significantly on the surrounding tissues

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Figure 51: Temporal profile of a THz pulse reflected from the sandwich
structure, overlapped with digital filters. (Reprinted from the Ref. [289])

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Figure 52: A multi-temporal THz image of a sandwich structure comprised
of three metal discs located between cardboard layers. (Reprinted from the
Ref. [289]) AN
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Figure 53: Ex vivo images of a human skin sample with BCC: (a) a histolog-
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ical cross-section; (b) a multi-temporal THz image; (c) a multi-spectral THz

image. (Reprinted from the Ref. [289])
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and the position of the tumor. Most of the techniques proposed to date,
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analyze the temporal shape of a reflected pulse rather than its spectrum.
Since a THz pulse broadens when reflected from a lesion in comparison with
that reflected from a healthy tissue, by using specially adjusted time-domain
digital filters, one can ensure a clear distinction between areas of healthy and
pathological tissues; Fig. 53 (b).
In general, the concepts of multi-spectral regions and multi-temporal do-

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mains in THz imaging have been tested with the aim of improving image
contrast for the detection and localization of substances of interest and hid-
den objects. Efficiency of multi-spectral and multi-temporal THz imaging

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of solid and bulk materials, sandwich structures and biological tissues was
successfully demonstrated.

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9 Methods for processing and analyzing the
terahertz spectral data

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9.1 Chemical imaging in the terahertz range
Usually analysis of multidimensional spectral data will include the fol-

U
lowing stages:

• preprocessing; AN
• selection of the informative features and reduction in the dimensions
of the feature space;
M
• development of the decision rules.
D

Chemical imaging combines spectroscopy and imaging; it can reveal the

distribution of various molecular substances in a biological liquid or tissue.
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The mechanism of the THz radiation absorption in biological tissues is

still at the stage of discussion, but intra- and intermolecular interactions are
known to play an important role. The THz range also contains absorption
bands associated with rotation and low-frequency vibration modes of biolog-
EP

ical macromolecules, deformations of hydrogen bonds; these are often called

”Structural Fingerprints”. Currently, the THz absorption spectra of pep-
tides, proteins, nucleotides and DNA, amino acids, including glycine, leucine,
C

cysteine, balanine, enantiomers (D- and L-alanine), were studied [297, 298].
Saccharides are considered to be one of the most important biomolecules.
AC

The spectra of glucose, fructose, mannose, galactose and sucrose in polycrys-

talline and amorphous forms were investigated using the THz-TDS [299].
Polar molecules have rich absorption bands in the THz range. In partic-
ular, far-IR absorption vibrations bands of water molecules caused by com-
binations of the symmetric and asymmetric stretches, and bending of the
covalent bonds. In solutions, water hydrogen bonds are much weaker than

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the covalent intramolecular bonds, as their bond lengths are much longer.
Steric effects from dipole moments in water clusters varying according to
hydrogen distance are appeared in ro-vibrational modes shifts at THz fre-

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quencies. These shifts are correlated with water molecules potential, which
indirectly influences on the interaction with the surrounding molecules, in
particular, on hydration shell formation in proteins [300].

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Unlike IR absorption and Raman spectroscopy techniques, which are sen-
sitive to femtosecond-scale intramolecular dynamics – i.e., bond vibrations,
the THz spectroscopy allows one to analyze the intermolecular dynamics –

SC
i.e., molecular rotations associated with hydrogen bond breaking. But, the
identification of pure compounds using THz-TDS systems based on molec-
ular signatures is still not straightforward because of the inherently broad
spectral signatures in liquids.

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Nevertheless, sophisticated methods of THz spectra analysis, based on the
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pattern recognition approaches and the databases of biomolecules spectral
signatures, give a chance for the chemical imaging realization in the THz
range. Currently, a growing number of multiply confirmed observations of
particular resonant signatures, that may be attributed to the presence of
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many compounds in a pure form, is appearing. A compilation of readily
identifiable spectral signatures of biomolecules has already been created by
researchers at Durham University [300]. There are other THz spectroscopic
D

databases, such as the THz database 2.0, which was created by the Tera-
photonics Laboratory, RIKEN Sendai [301], and the THz database released
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by NICT, RIKEN and Tohoku University [302].

9.2 Preprocessing of terahertz waveforms

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One of the main challenges of the THz imaging is associated with improve-
ment of the signal-to-noise-ratio in the the experimental data. Averaging of
the multiple measurements in the THz-TDS (see section 3.1) yields improve-
C

ment in the signal-to-noise-ratio (SNR) per pixel. This SNR improvement is

proportional to the square root of the number of co-averaged time-domain
AC

scans [300]. Evidently, this approach is time consuming, especially in case of

2D or 3D measurements.
Currently, there are a lot of digital filters have been proposed for sufficient
signal de-noising. The Gauss filter is a popular one, which allows for smooth-
ing the temporal shape of THz-TDS waveform, thus, providing a decrease
in the high-frequency noise [303]. Various window filtering approaches are

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applied for improving the quality of the THz data; among them: the wavelet-
domain de-noising technique [304, 305], the time-domain apodization – i.e.,
the window filtering, which reduces the frequency-domain Gibbs noises, orig-

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inating due to the edge effects [300], recursive filters [306], etc.

9.3 Feature extraction methods

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Each THz spectrum contains information about the molecular compo-
nents in a sample and can be considered as a vector in a feature space.

SC
Pattern-recognition-based techniques classification for groups under study is
based on separability of them in feature space and provides probabilistic dis-
crimination of biomarker profiles, which can include, for example, chemical
compounds. Notably, this approach does not need identification of individual

U
biomarkers, but is based on probabilistic recognition.
The problem of weak distinguishability of feature vectors in high dimen-
AN
sion feature space is known. The essence of this problem is that, when the
dimension of the feature vector increases, the amount of data, necessary for
the correct classifier training, grows exponentially. This is due to the fact
M
that, according to the central limit theorem, the probability of a normal dis-
tribution of two random vectors tends to unity with increasing of dimension
of these vectors, that is, the difference between them is neglectable [307]. To
D

solve this problem, one must provide separation of informative features of

the object state. This simultaneously provides identification of the hidden
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structure in the data and, in some cases, the noise reduction.

The THz-TDS features, which are suitable for classification, are repre-
sented by the height, the shape, and the delay of the THz pulse in the time
domain, as well as the spectral content of the THz pulse in the frequency
EP

domain. The medium parameters, as features, include the complex dielec-

tric permittivity (or the complex refraction index), differential transmittance
and absorbance. Essentially, features should consist of parameters that dis-
C

play non-transformed structural characteristics: moments, power, amplitude

information, energy, etc., as well as transformed structural characteristics:
AC

frequency and amplitude spectra, coefficients of the wavelet decomposition,

coefficients derived from the corresponding time series, etc. [300].
There are continuous and discrete methods of separation of informative
features [308]. Discrete methods include so-called filters, i.e., algorithms
based on the selection of a subset from the original set of characteristics
(Pearson’s criterion, mutual information based on the Shannon information

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criterion and the Kulbak-Lebler divergence). Among the discrete methods,

there are methods of ”wrappers” (the classifier is considered as a black box
with the input of the generated feature sets, and the result of classification

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is evaluated) and ”built-in” methods that optimize the ”wrappers”-approach
in order to reduce the number of repeated classifications.
Continuous methods include the frequently used method of Principal

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Component Analysis (PCA), Factor Analysis, Isomap, Diffusion Maps, Mul-
tilayer Autoencoders, etc.
The basic idea of PCA is to find the reduced number of new variables

SC
which are enough for recovery of the initial variables, possibly with insignif-
icant errors. PCA transforms correlated variables into a lower number of
uncorrelated variables called principal components. Principal components
are ordered such that first components describe largest variance in compari-

U
son with the next components. In general, when the cumulative variance of
AN
the first principal components becomes large enough (typically 75-85%), the
other principal components can be neglected.
The mathematical background of PCA consists in decomposition of initial
experimental data 2D-dimension matrix X(I ×J) in a form of matrix product
M
[309]
X = T · P t + E, (57)
where T , P , E are the scores, loadings and residuals matrixes, respectively.
D

The loadings matrix contains weight coefficients which characterize the con-
tribution of initial features to the definite principal component. Scores matrix
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contains coordinates of the samples in the space of principal components.

The component analysis of complex media can be realized using the
Canonical Correlation Analysis (CCA). The CCA enables to identify and
EP

to evaluate associations between two sets of variables. The aim of CCA is

to maximize connection between the low-dimensional projections of the two
data sets, for example, between vectors of random quantities. The mathemat-
C

ical problem here is to find the closest fit approximation of the experimental
spectrum V by linear combination of individual molecular components U
AC

spectra set Xi
U= (58)
X
αi Xi ,
i

here, αi is an unknown concentrations of the specific components Xi con-

tained in complex medium. The criterion of the best approximation of the
vector by a linear combination is the maximum of the Pearson correlation

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Figure 54: THz absorption spectra corresponding to the saliva exosomes sam-
ples of a patient with colorectal cancer (left panel) and a healthy volunteer

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(right panel); here, the CCA-based approximation of the experimental data
is shown by the solid lines. (Reprinted from the Ref. [310])
AN
coefficient
cov(U, V)
(59)
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,
σU σV
here, cov stands for the covariance; σ is the standard deviation.
The study of exosomes of saliva and blood plasma by the THz-TDS tech-
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nique using CCA was carried out in Ref. [310]. Exosomes were sampled from
patients with colorectal cancer (n = 6) and from healthy volunteers (n = 5).
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The exosomes were characterized using flow cytometry on the content of the
most common exosomal membrane proteins: CD9, CD63, CD81, CD24. The
absorption spectra was measured in the frequency range of 0.3 to 3.0 THz
EP

using the THz-TDS (EKSPLA, Estonia). A substantive examination of the

sample absorption spectra was performed using the CCA method. The ab-
sorption spectra were taken from the RIKEN Sendai THz database [301].
The results of an approximation of the experimental spectra are presented
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in Fig. 54.
The presence of Glycine, L-Alanine, Mannose was revealed in all samples.
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The content of Mannose was less in samples with exosomes corresponding to

colorectal cancer than in samples with exosomes corresponding to healthy
volunteers.

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Figure 55: Supervised and non supervised classification methods.
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(Reprinted from URL: http://www.techsparks.co.in/hot-topic-for-project-
and-thesis-machine-learning/ October 10, 2018)
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9.4 Pattern classification
In most medical applications, the diagnostics consist of making a decision
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based on the specific data about the object (feature values) and possible clas-
sification outcomes. Pattern classification provides the separation of profiles
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of feature vectors into predefined classes, and then the assignment of each
pattern to a particular class. Thus, pattern classification is a powerful tool
for intellectual analysis of medical data (Intelligent data analysis, IDA). IDA
is mainly aimed at extracting knowledge and patterns contained in primary
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medical data.
There are supervised and non-supervised classification methods; for ex-
ample, see Fig. 55. Supervised learning means training the classifier when
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the certain set of vectors is available for which belonging to one of the classes
is known. To train the classifier, only a part of the data should be used
AC

(training sample).
When classifiers are trained, there is a danger that it will be too well ad-
justed for training data, which will lead to the difficulty of correctly classify-
ing new (unseen) data. This problem is called ”overtraining” or ”overfitting”
of the classifier. Estimation of the quality of the resulting classifier, using a
test on the training data, may lead to the fact that retraining (if it exists)

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may not be detected.

A more adequate estimation is the evaluation of performance on a test
suite-a set of data classified by class, but not used in the training process.

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This can include both a cross-validation and an external validation to avoid
discrepancy. In the n-fold cross-validation the dataset is randomly divided
into n subsets of equal size and after that n − 1 subsets are used for training

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and the remaining subset is used for examination of classification quality.
This procedure should be repeated until all n subsets have been used as test
set [311, 312]. The limit case of this algorithm is ”leave one out of the cross-

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validation”, which corresponds to n being equal to the experimental data set
size. In the external validation, new dataset obtained by repetition of the
measurements with the same population is used [313].
The Support Vector Machine (SVM) is a frequently used supervised

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method. SVM binary classification is based on building up the maximum-
AN
width stripe, which separates groups under study. The algorithm is based
on scalar product of the feature vector analysis. When the building of such
stripe is impossible, the kernel trick can help to provide classification, using
scalar product of the feature vector functions. The application of SVM to
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the problem of data classification is realized by a training set with objects
that belong to one of the two classes; each new object is assigned to one of
these classes. The problem is defined as follows
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(x1 , y1 ), . . . , (xm , ym ) ∈ X × {±1}, (60)

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where X is a nonempty set; m is the number of objects in the training set;

yi are so-called labels; xi are the objects under classification. Each classified
object is a vector in n-dimensional space.
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Thus, there is the task of some classifier rule building

n
! !
a (x) = sign wj x − b ≡ sign hw, xi − b ,
j
(61)
X
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j=1

where operation hw, xi defines the scalar product of vectors; the vector w =
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(w1 , w2 , . . . , wn ) ∈ Rn and the scalar threshold b ∈ R are the algorithm

parameters.
The SVM based on the THz absorption spectra as feature vectors was
successfully used for identification of seven pure illicit drugs, and for the
discrimination between normal and malignant tissues of the breast [299].
The set of substances was indentified by using THz images [314]. Although

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the samples have similar optical properties, their complex attenuation at the
THz range is significantly different due to differences in the spectral shape of
extinction coefficient, refractive index and scattering properties. The authors

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provided comparison of four different machine learning classifiers: multi-
nomial logistic regression with ridge estimators (MLR) classifier, k-nearest
neighbors (KNN), SVM and naive Bayes (NB). For the same number of

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feature vectors, the accuracy of MLR, KNN, SVM, and NB classifiers was
88.24%, 90.19%, 74.51%, and 56.86%, respectively.
Recently, Extreme Learning Machines (ELMs) classifiers have been de-

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veloped, providing the lowest training errors among the machine learning al-
gorithms and, in particular, the SVM classifier. Furthermore, ELM permits
us to classify the inputs represented by the linear complex-valued feature
vectors; thus, preserving the information about the phase of the THz sig-

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nal. The approach is based on concepts developed for quaternary variables
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classification through the introduction of two complex-coupled hyper-planes.
A classification of powdered samples based on their THz spectral signatures
was performed in order to demonstrate the advantages of the complex-valued
ELM classifiers over the SVM [315].
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The Mahalanobis distance classifier is a type of minimum distance classi-
fier, which is optimal for normally distributed classes with equal covariance
matrices (linear discriminant) and equal a priori probabilities. Such classifier
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is often chosen because of its simplicity. It provides reasonable results for a

variety of biomedical waveforms [300].
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In case when the THz spectra posses small differences in the shape, it
is difficult to use them for identification of the samples. In Ref. [299], the
fuzzy pattern recognition method was introduced in order to identify the
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THz spectra as follows. If we have m subsets Ai , i = 1, m of the universe

U = (u1 , u2 , . . . , un ), then a predictive fuzzy subset B of the universe U is
m
δ(B, U ) = δ(B, Ak ), (62)
_
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k=1
where δ is the measure of the proximity of B and U . In the case of the Euclid
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distance usage as the measure of the proximity, the Eq. (62) takes the form
v
u1 X
u m
δ(A, B) = 1−t (µ A (ui ) − µB (ui ))2 , (63)
n i=1
where A, B are two fuzzy subsets of U ,µA (ui ) ∈ [0, 1] is the membership
degree of ui in A.

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Some typical amino acid and saccharide biomolecular samples were inves-
tigated using PCA and fuzzy pattern recognition applied to the THz trans-
mission spectra, in order to prove the feasibility of this approach. Firstly,

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PCA is applied to reduce the dimensionality of the original spectrum data
and extract its features. Then, instead of the original spectrum variables,
the selected principal component score matrix was used in the model of fuzzy

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pattern recognition, where a principle of fuzzy closeness is employed to iden-
tify those samples. The observed results demonstrate that THz spectroscopy
combined with PCA and fuzzy pattern recognition can be efficiently utilized

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for automatic identification of biomolecules. The proposed approach pro-
vides a new effective method of detection and identification of biomolecules
using the THz spectroscopy [299].
Thereby, the methods of processing and analyzing of the THz spectral

U
data are of great importance. The mechanism of THz wave absorption in
AN
biological tissues remains a topic of discussion, but the intra- and inter-
molecular interactions are known to play an important role. Furthermore,
the frequency-dependent THz dielectric response of a medium contains ab-
sorption bands associated with rotation and low-frequency vibration modes
M
of biological macromolecules, deformations of hydrogen bonds. Identification
of pure compounds using molecular signatures in the THz range is still not
straightforward because of the inherently broad spectral signatures. Nev-
D

ertheless, numerous abovementioned approaches for solving the problems

of substances’ identification and biomoleculs’ detection have been recently
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proposed, promising novel applications of THz technology in biology and

medicine.
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10 Heat transfer in tissue irradiated by tera-

hertz radiation
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At standard atmospheric pressure the dielectric permittivity ε(ν, T ) =

ε0 (ν, T ) + i.ε00 (ν, T ) of pure water depends upon the frequency ν and the
AC

temperature T . In Ref. [19], Ellison et al. established an accurate interpola-

tion function for the complex permittivity of water. This data was obtained
in the temperature range of 0 ◦ C to 100 ◦ C and the frequency range up to
25 THz. The three Debye relaxation methodology was found to be the most
accurate for this purpose. An expression for the absorption coefficient can

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be written as a function of real and imaginary part of the complex dielectric

permittivity [316]

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q 1/2
4πν  ε0 (ν, T0 )2 + ε00 (ν, T0 )2 ε0 (ν, T0 )2 
α(ν) = − , (64)
c0 2 2

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where c0 is the speed of light in a free space; T0 is the initial temperature.
As a sample of water in the form of a disk is exposed to THz radiation
focused into its center, then, the two main effects are observed [316]: THz

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radiation heats the top layer of the water disc and the heating efficiency is
about 40%, when the frequency is varied from 0.1 to 10.0 THz. Dielectric
properties of liquid water and ice have been investigated as a function of
temperature in the range of 253 to 353 K [317]. Water demonstrates a de-

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crease in the imaginary part of the dielectric permittivity in the range of
AN
0.5 to 2.5 THz. The same trend is observing for solutions of proteins, such
as human serum albumin, in a water buffer over the entire frequency range
of 0.01 to 3.00 THz [318]. When heating the sample from 20 to 70 ◦ C, the
absorption coefficient and the refractive index at THz frequencies increase.
M
This process can be explained not only by the change of the protein chains
with the water molecules, but also by the structural changes in protein dur-
ing heating. Such a change in THz absorption can be used to estimate the
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temperature increase of the sample containing water. The thermal diffusiv-

ity of water at 10 ◦ C is 0.00138 cm2 s−1 . The measured thermal diffusivity
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for some proteins has been found to be equivalent to those for water – i.e.,
0.00130 cm2 s−1 [319].
Dermal fibroblasts were irradiated by the THz radiation at the frequency
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of 2.52 THz; the irradiance was 84.8 mW/cm2 and the exposure time was
80 min [320, 321]. An increase in the temperature of cells was measured
by a thermocouple and an infrared camera, as well as modeled using the
finite-difference time-domain (FDTD) method. The latter is widely applied
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to simulate the heat transport through the two-dimensional homogeneous

model of cell media. The temperature of the irradiated sample was shown to
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demonstrate a sharp increase during the first 3 min, while the experimental
and numerically estimated temperatures were close to each other.
The universal thermal model is obviously need to be obtained for esti-
mating the heating of biological object irradiated by either CW or pulsed
THz sources. For this estimation, the thermo-physical properties of the an-
imal eye cornea were taken from the Ref. [322], where the average value of

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the thermal conductivity is η = 0.32 W/mK; the heat capacity of cornea is

C = 3158 J/kgK; the thickness of the sample layer is h = 0.7 mm; the power
of THz radiation was varied in the range of ∼ 1 nW to ∼ 1 W; the ambient

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temperature was T =25 ◦ C. The numerical simulation was carried out using
MathCad software for the one homogeneous porcine cornea layer. As a re-
sult, a histogram, summarizing the data on heating of the sample irradiated

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by THz sources featuring various output power, was built (see Fig. 57).
The heat transport was described by Eq.
dT (x, t) d2 T (x, t)

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Cρ =η + Q (x, t) , (65)
dt d2 t
where ρ is the object density; T is the temperature; t is the THz exposure
time; and x is the coordinate. The function Q (x, t) describes the density

U
of thermal sources. In other words, it is an amount of heat for a period
of irradiation time. The Eq. (65) represents an equality of changes of the
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energy of a matter in a unit of volume, a sum of the thermal flux density,
and the thermal sources per volume unit. The density of thermal sources is
a function of spatial coordinatex and time t
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Q (x, t) = q (t) e−αx , (66)
here, α is the absorption coefficient; q is the initial density of thermal sources.
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Any even function, which satisfies the Fourier conditions, can be consid-
ered as a sum of cosine harmonics and a constant
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q (t) = q̄ + qn cos (nωt) ≡ q̄ + qe (t) , (67)

X

where n is an integer; ω is the angular frequency of the field. Such a sum

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of the cosine harmonics describes fluctuation of the function qe around an

average power density q̄; see Fig. 56.
Furthermore, we can consider the temperature function as the sum
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T (x, t) = T̄ (x) + Te (x, t) , (68)

AC

where
Te (x, t) = Tn (x) cos (nωt) + θn sin (nωt) . (69)
X

n
Thus, the Eq. (65) can be rewritten as
dTe (x, t) d2 Te (x, t)
Cρ =η + qe (t) e−αx . (70)
dt dx2
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The boundary conditions are:

dTe (x, t)
η |x=0 = β Te (x, t) |x=0 , (71)

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dx
dTe (x, t)
−η |x=h = β Te (x, t) |x=h , (72)
dx

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where β is the convection heat transfer coefficient. By substituting the series
in Eq. (70) with the boundary conditions from Eqs. (71) and (72), we obtain
a system of equations with unknown Tn and θn represented by the sum of

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general and special solutions
x x x x
Tn = A1n eγ1 + A2n eγ2 + A3n eγ3 + A4n eγ4 , (73)

U
x x x x
θn = Bn1 eγ1 + Bn2 eγ2 + Bn3 eγ3 + Bn4 eγ4 , (74)
where γ is the complex number
√
2
AN √ !s
2 nω
γ1,2,3,4 =± ±i . (75)
2 2 η
M
Next, the coefficients before exponents were calculated; thus, leading to
an Eq. for the average temperature
D

q̄eαx
T̄ (x) = − + Kx + L, (76)
ηα2
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where      
q̄ −αh
α
e 1
ηα
− 1
β
+ q̄h
ηα
1+ β
ηα
+ q̄
α
1
ηα
+ 1
β
L = T0 + , (77)
2+ βh
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η
C
AC

Figure 56: A graph illustrating the average radiation density q̄ and the fluc-
tuation of the function, q,
e described by the sum of cosine harmonics; see
Eq. (67). (Courtesy of O.A. Smolyanskaya)

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q̄β βL βT0 q̄
K=− 2 2
+ − − . (78)
η α η η ηα
The result of the numerical modeling indicates that exposure of tissues

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to THz radiation with the average power of 100 µW, 10 mW, 100 mW, and
300 mW heats the object by 0.004, 0.43 4.32, and 12.95 ◦ C, respectively.
The elevetion of the tissue layer temperature were found to be noticable

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when the power reaches a few milliwatts. The calculated histogram for the
tissue heating using the THz sources with different average output power is
presented in Fig. 57. The described thermal model can be used for evaluation

SC
of a tissue heating by THz source operating in either pulsed or CW regimes.

11 Tissue optical clearing in the terahertz range

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THz radiation is sensitive to free and bound water content in tissues.
AN
A hyperosmotic immersion agent, e.g. glycerol, includes a flow of free and
weakly bound water out of a tissue and diffuses inside via substitution of
free water in the inter- and intra-cellular space. These two facts could help
M
to enhance the THz-wave penetration depth, increase the contrast of THz
images, as well as expand the diagnostic capabilities related to bound water
properties.
D

In normal tissue, proteins are able to form a hydration shell [14, 15] that
reduce a mobility of water molecules. In the malignant tumor tissues, pro-
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tein molecules demonstrates the reduced ability to bind neighboring water

molecules and these tissues content more free water than bound one. Ap-
plication of hyperosmotic immersion agents induces the temporary removal
of free water from tissue, and the ratio of free-to-bound water should be de-
EP

creased. This is a way to control balance between free and bound water in
soft tissues, the kinetics of which is important to study to provide optimal
immersion optical clearing. However, the investigations of kinetics of optical
C

clearing for normal and pathological tissues has not yet been done in the
THz frequency range.
AC

It is worth mentioning that the optical clearing technique is an easy and

low-cost approach for improving contrast and the spatial resolution of optical
diagnostic methods [7, 9, 149, 327–330]. It reduces the light scattering in
tissue, enhance the light focusing ability and provides a greater penetration
depth. Many of optical imaging methods in the visible/NIR range are used
together with an an optical clearing technique. In laser surgery and therapy,

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AN
Figure 57: An increase of the tissue temperature caused by its exposure to
THz radiation. Here, ∆T = 0 ◦ C corresponds to initial temperature of T =
M
25 ◦ C; TDS stands for a THz time-domain spectrometer [118]; PCA stands
for a THz photoconductive antenna [128]; OR - stands for an optical recti-
fication method [323]; CO2 stands for an CO2 laser frequency mixing [323];
D

PM - stands for photomixers [128]; DFG - stands for a difference-frequency

generation in nonlinear crystals method [323]; OPL - stands for optically
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pumped laser method [323]; GL – stands for a gas laser [118]; RL – stands
for a Rydberg laser [324]; D - stands for a diod lasers [118, 325, 326]; p-Ge -
stands for a p-Ge lasers [118, 323]; EA - stands for a Electron Accelerators
EP

method [118]. (Courtesy of O.A. Smolyanskaya)

optical clearing helps to keep a sharp focus, reduces the radiant exposure
C

and decreases the light beam distortion.

Dehydration is a physical process involving the loss of water by tissue; it
AC

is one of the ways to achieve optical clearing. Nowadays several methods of

optical clearing are well understood [7,9,149,327–330]. One of them is tissue
immersion method involving the use hypersmotic chemical agents. Among
the most commonly used agents are glycerol, polyethylene glycol, glucose,
and many others [9, 149, 327]. Specified agents are promising as they are
biocompatible, nontoxic, and characterized by their high diffusion rate and

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dehydration ability.
Mechanical compression, freezing, heating, gelatin or paraffin embedding,
or lyophylization of soft tissue samples also belong to the promising group

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of optical clearing methods [7–9, 149, 226,229, 258, 259,265, 266, 269,327, 331].
These methods also cause tissue dehydration by a varying degree depending
on the agent used. However, for example, conventional freezing could initi-

RI
ate ice crystal formation in the intra- and extra-cellular spaces and destroy
tissue structure. Lyophilization is a method of snap freezing of a tissue at
low-pressure and low-temperature conditions that causes water to sublimate,

SC
while the structure and molecular integrity are retained; this method removes
up to 90–99% water. The lyophilized, frozen or gelatin/paraffin embedded
tissue samples provide measurement reproducibility over an extended period,
and they are also easy to handle and measure [259, 265, 268]. Unfortunately,

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these methods cannot be used in in vivo conditions.
AN
Compression (squeezing) or stretching of a soft tissue may also cause
temporal displacement of free water in the interstitial liquid and blood out
of compression site and also provide a more close packing of tissue compo-
nents [8, 149, 327, 332, 333]. Fortunately, this technique is applicable in vivo,
M
but specific instrumentation should be designed to provide an appropriate
mechanical action [334].
The immersion optical clearing technique was successfully used for differ-
D

ent soft tissues like skin, muscle, eye sclera, and others, as well as for hard
tissue like bones and cartilage or for blood in vessels [149,327]. In case of hard
TE

tissue, their high porosity plays the main role in the immersion agent distri-
bution. In blood, dextran, glucose, X-ray contrasts, and free hemoglobin are
used as optical clearing agents.
EP

Typically, two major diffusion fluxes between the tissue and the enviro-
ment would be generated under the action of hyperosmotic biocompatible
immersion agent surrounding tissue layer. First one is the water flux di-
rected from tissue and the second one is the applied agent flux directed into
C

the tissue. Therefore, two main processes can be observed: the dehydration
of tissue and refractive index matching between tissue components due to
AC

replacement of free water in the interstitial space by molecules of the agent

that presents higher refractive index than water.
A one-dimensional free diffusion equation for water or an immersion agent
transport has the form
dCf (x, t) d2 Cf (x, t)
= Df , (79)
dt dx2
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where Cf (x, t) is the fluid concentration [g/ml]; Df is the water/agent diffu-

sion coefficient [cm2 /sec]; t is the time of diffusion [sec]; and x is the spatial
coordinate along the sample depth [cm].

PT
In the first-order approximation, a solution of the diffusion Eq. (79) for
the volume-averaged concentration of an agent Ca (t) has the form [149,327]:

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d
1Z t
  
Ca (t) = Ca (x, t) dx ∼
= Ca0 1 − exp − , (80)
2 τ
0

SC
where τ is the characteristic diffusion time. If the diffusion coefficient Df
of water or chemical agent in a tissue is known, it is possible to define the
time-dependent concentration of water or agent at depth x within a tissue

U
sample. The formula for the diffusion coefficient Df in the case of the agent
application to one side of a plane tissue sample with the thickness d has the
form AN
Df = d2 /τf , (81)
where τf stands for the characteristic diffusion time for the molecular flows
M
(water or agent).
If the hyperosmotic agent is applied, more fluid (free water) leaves the
tissue than agent enters the tissue because the tissue matrix and the cell
D

membrane permeability is different for the agent and water. Therefore, tis-
sue dehydration occurs due to not only free water leaving, but also because
TE

of collagen hydration shell and water hydrogen bonds degradation at later

stages. As a result, tissue shrinkage occurs due to the much better pack-
ing of tissue components (fibrils); the tissue becomes more dense with less
thickness [327, 328]. Tissue thickness and volume depend on the initial ion
EP

hydrogen concentration (pH) of the tissue and acid or alkaline properties

of the immersion agent. At osmotic pressure on a soft tissue, similarly to
mechanical compression [8], bound water can be transformed into the free
C

water, and the free water will be directed outside the tissue; such exchange
of different states of water can be described by Eqs. (80) and (81) [10].
AC

Dehydration of tissue, the replacement of free water by the hyperosmotic

immersion agent and the increase of volume fraction of scatterers (collagen
fibrils) could reduce the light scattering due to refractive index matching of
scatterers and modified interstitial fluid with less amount of water and bigger
amount of the agent, as well as due to less free space between scatterers
and their better ordering. Kinetics of dehydration and agent replacement

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processes is defined by characteristic diffusion time τf or diffusion coefficient

Df [82]. Such kinetics is characteristic for tissue under study and agent
applied. In particular, this property allows one to differentiate healthy and

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pathological tissues.
It has been shown in Refs. [328,329] that compare to diffusion rate of hy-
perosmotic agent in healthy tissue, diffusion rate and correspondingly kinetics

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of optical collimated transmittance are changed strongly if some pathology
is present, such as diabetes or cancer. For example, with diabetes, pro-
tein glycation leads to a decrease tissue permeability for different molecules,

SC
including water and glucose [328]. From measured kinetics of optical trans-
mittance, the higher free water content in the cancerous colorectal mucosa
was found than in healthy mucosa, which demonstrated the possibility of
quantifying free and bound water in soft tissues [329].

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Immersion optical clearing technique is not widely used in combination
AN
with THz tissue investigation. Lyophilization is a convenient technique for
THz spectroscopy, as it preserves the molecular and structural properties
and THz radiation passes with an acceptable attenuation through the water-
free sample. In Ref. [331], Png et al. have reported that lyophilized tissue
M
samples, such as kidney, colon and stomach, retain their thickness and vol-
ume. The THz radiation transmitted through the lyophilized tissue sample
seems to be like nitrogen-dried necrotic tissue that indicates the protein de-
D

naturation during the lyophilization process. It was reported by Pickwell-

MacPherson et al. in Ref. [264] that the refractive index and the absorption
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coefficient of the muscle and adipose tissue were reduced during the formalin
replacing the normal fluid in cells with a gel like matter and dehydration of
the tissue occurs; see Fig. 58. The THz optical properties of fresh tissue were
EP

close to those of water. The fat cells in the adipose tissue contains much less
water – i.e., about 10–30%; therefore, the THz optical properties of adipose
tissues are not so dramatically reduced as for muscle tissue.
Optical clearing agents that have been validated for THz spectroscopy
C

and imaging are commonly referred to as THz penetration-enhancing agents

(THz-PEAs). The THz-PEA should have the absorption coefficient lower
AC

than that of water; for example, glycerol is a representative example of THz-

PEA, the absorption coefficient of which is three times smaller than that of
water at 1.0 THz. The evaluation of corneal hydration by using low concen-
trated polyethylene glycol (PEG) solutions has been described in Ref. [335].
The nine corneas were soaked in 0.15M NaCl and added 3%, 5%, and 7%
PEG solutions for 3 days and the hydration of the sample has varied from

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SC
Figure 58: THz refractive index (a) and absorption coefficient (b) of skeletal

U
muscle before and after the formalin fixation for 24, 48, and 72 h. Bulk water
and formalin optical properties are also plotted, as a reference. (Reprinted
from the Ref. [264])
AN
79.1% to 91.5% by mass. It has been revealed that the dependence of the THz
M
reflectivity on water concentration approximately linearly decreases with an
increase of frequency from 0.25 to 1.05 THz (see Fig. 59), and the same trend
was observed for the slope of these dependencies.
D

The change in the noise-equivalent water concentration sensitivity was

calculated as
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∆R
∆CH2 O = dR , (82)
dCH2 O

where CH2 O is the water concentration, and R is the THz reflectivity. The
EP

estimated hydration sensitivity of the THz spectroscopy is 0.19%. This hy-

dration level is much lower than that provided by the standard ultrasound
and pachymetry methods of the corneal diagnostics.
C

In Ref. [266], Oh et al. have verified the glycerol as a THz-PEA in the

diagnosis of cancer. In Fig. 60, we illustrate an improvement in the THz
AC

images of an artificial tumor in the rat skin sample under the influence of
glycerol. First, the matrigel drops, that served as an artificial tumor, have
been placed onto the optically cleared and non-cleared samples of the skin;
see Fig. 60 (a). If the whole THz-TDS signal was processed, no differences
were found; see Fig. 60 (b). When the main peak-to-peak was not accounted
for in the processing, the artificial tumor was well observed in the THz images

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only in the case with applied glycerol; see Fig. 60 (c).

In Refs. [336–338], Kolesnikov et al. have examined the glycerol, PEG
and propylene glycol (PG), as THz PEAs. These agents demonstrate similar

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characteristics, such as signal amplitude and phase, in the frequency range
0.2 to 1.6 THz; see Fig. 61. In comparison to water, the agent reflection signal
amplitude essentially differs; thus, the chosen agents satisfy the requirements

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of tissue optical clearing in the THz domain. In the low-frequency region,
the phase of the signal for water experienced the characteristic curve that is
a marker of water in a tissue.

SC
As it was expected, the immersion agents decrease the refraction index
and the absorption coefficient in the THz band for muscle, and these spectra
decay with time; see Fig. 62. It is interesting to compare clearing abilities of
these agents with formalin [264]; see Fig. 58. One could notice that tradition-

U
ally used optical clearing agents, such as glycerol, PEG and PG [149, 327],
AN
are much more efficient than formalin because only 8–10 min is required to
make muscle tissue more than 2–5 fold less absorptive in the frequency range
of 0.2 to 1.0 THz. The additional benefit for a better THz radiation trans-
mittance is due to the decrease of the refractive index from 3.1–2.6 to 2.1–1.9
M
in the 0.2–1.0 THz range (see Fig. 62 (b)); thus, the Fresnel reflection from
the tissue–air interface will be significantly less with optical clearing. The
average diffusion coefficient Df has been calculated using Eq. (81), where
D
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C EP
AC

Figure 59: THz reflectivity versus corneal water concentration at different

THz frequencies. (Reprinted from the Ref. [335])

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Figure 60: THz imaging of artificial tumors placed on tissue samples without
and with glycerol impregnation: (a) top views of the tissue samples and the
AN
artificial tumor; (b),(c) THz images of the tissue samples with the artificial
tumor, when the peak-to-peak values of the whole THz-TDS signal was used
and when the main peak-to-peak range was cut off, respectively. The size of
the THz image was 5 × 3 cm2 . (Reprinted from the Ref. [266])
M
D
TE
C EP

Figure 61: ATR spectrum of the amplitude (a) and phase (b) for the distilled
water and some THz-PEAs. (Reprinted from the Ref. [336])
AC

the characteristic diffusion time for the molecular flows (water/agent) τf was
determined from the experimental kinetic curves for particular tissue thick-
ness d; see Fig. 63. At the characteristic diffusion time τf , the kinetic curves
become saturated. It was found that optical clearing efficiency of glycerol

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Figure 62: THz absorption coefficient (a) and refractive index (b) of the
bovine muscle tissue impregnated by glycerol. The time corresponds to the

U
cumulative time of holding the sample in the agent solution. (Reprinted from
the Ref. [336])
AN
and PEG is better than that of PG.
When dehydrating agents 100% glycerol, 40% water glucose solution,
M
PEG-600, or PG were applied to the normal and cancerous rat skin, PEG-600
demonstrates most effective results for the THz optical clearing [336–338].
The optical clearing of cancerous tissue occurs more faster than those for
D

normal tissue; moreover, optical clearing is faster for the in vivo studies (13–
17 min), as compared to the experiments in vitro (20–80 min). However, in
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in vivo conditions, the subsequent rehydration process takes place because

of physiological response of living tissue on dehydration and osmotic stress.
In Ref. [339], Smolyanskaya et al. have demonstrated that a deeper pen-
EP

etration of THz waves into the mouse skin in vitro treated with the glycerol
can be related with the decrease of free-to-bound water ratio in a sample.
The calculated data obtained experimentally by the nuclear magnetic reso-
nance (NMR) method has demonstrated that the total water in % and the
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hydration ratio (H) of the mouse skin sample decreased with the glycerol
AC

treatment. The better results have been achieved at 70% and 100% of glyc-
erol concentrations, such as the total water reduced by 28.8% and 25.3%,
respectively.
Yamaguchi et al. have proposed the following equation to estimate water

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(a) (b)

Figure 63: Kinetics of the THz signal reflected from the muscle tissue: (a) an

U
action of PEG and PG; (b) an action of glycerol for two tissue sample thick-
nesses. (Reprinted from the Ref. [336])
AN
content in the measurement site of a tissue [244]
M
A (100 − A)
ntissue = nwater + nother , (83)
100 100
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here, ntissue , nwater , and nother stand for the refractive indices of tissue, water
and other biological components in a tissue, respectively; A is the percentage
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of water in the volume, where the THz wave interacts with a tissue. If water
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(a) (b)
Figure 64: THz refractive index of normal and tumor rat brain tissue:
(a) fresh tissue; (b) paraffin-embedded tissue. (Reprinted from the Ref. [244])

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in a tissue sample is substituted by paraffin, then nwater in Eq. (83) would

be substituted by refractive index of paraffin nparaffin . In this article, the per-
centage of water in a tissue has been estimated using the acquired value of

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nparaffin and by subtracting ntissue−paraffin from ntissue . The estimated percent-
age of water A is approximately 5% higher for tumor tissue, as compared to
normal ones. In this method, the fresh and paraffin-embedded normal and

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tumor brain tissues have been studied; see Fig. 64. It has been shown that
the higher refractive index in tumor tissue is connected not only with the in-
crease in water content, but also with approximately 15% increase of the cell

SC
nucleus size in tumor tissue. Almost the same results have been reported for
renal normal and tumor tissues in Ref. [340]. As shown in Fig. 65, the optical
properties of fresh normal and tumor tissue of the brain are similar to each
other; the refractive index at 1.0 THz is about 2.2–2.1 [244]). The refractive

U
index is dramatically decreased under the action of dehydration process in
AN
both normal and tumor tissue; it achieves values of 1.5 for the normal tissue
and 1.8 for the tumor one. Thus, the dehydration process leads to a higher
difference in the THz optical properties of normal and tumor tissues.
Further experiments will be directed to the enhancement of biophysi-
M
cal and mathematical methodology connecting interaction between normal /
pathological tissues and immersion agents for improving the THz biomedical
imaging. The precise and safe concentration of immersion agent is needed
D

to be defined to be safely applicable to living tissues in vivo. Many soft and

hard tissues and body fluids remain unstudied in the framework of optical
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clearing for more effective THz diagnostic and therapeutic applications. The
ratio of volume fractions of tissue components, including cell nuclei and cy-
toplasm, as well as the alterations of real and imaginary parts of the THz
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dielectric permittivity and the Fresnel reflection at the interfaces inside the
tissue under the action of immersion agents are also required to be clarified.
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12 Tissue-mimicking phantoms in the tera-

hertz frequency range
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12.1 Introduction
Over the past decades, tissue-equivalent phantoms have been widely em-
ployed in a broad range of electromagnetic wave frequencies, spanning the
band between the X-rays and the radio waves. They mimic the optical prop-

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erties of human and animal soft and hard tissues, as well as cultured cells
and blood. The education and research activities in the field of biophoton-
ics often requires such test objects due to lack of equipment and facilities

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necessary for work with living animals. Optical phantoms are designed to
provide periodic calibration, validation, an quality assurance for new medical
optical devices. The phantoms should possess long term stability in order

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to be able to examine the device performance in time. A limited number of
investigations of phantoms in the THz band have been conducted [341–350].
Four main tendencies in the development of tissue phantoms for the THz

SC
applications have been evident over the last fifteen years:
• phantoms revealing the composition of tissue;

U
• phantoms imitating the degree of tissue hydration;
• phantoms of healthy tissues and tumors;
AN
M
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Figure 65: THz absorption coefficient (left panel) and refractive index
(right panel) of normal and tumor renal tissue, both fresh and dehydrated.
(Reprinted from the Ref. [340])

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• phantoms of the skin for studying the drug delivery.

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12.2 Phantoms revealing the composition of tissues
The significant practical interest lies in the area of the application of THz
phantoms for analyzing the concentration-dependent effects in tissues. Based

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(a)
M
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(b) (c)
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Figure 66: (a) A content of water, lipids, and proteins in 4 types of healthy
human tissue – adipose, muscle (cardiac and skeletal), and skin; (b) a content
AC

of water, lipids and proteins in healthy tissues (muscle, mucosa and adipose),
and 3 diseased tissues (cancerous, dysplastic, inflamed), determined using
the spectrally averaged dielectric coefficient approach; (c) a content of water,
lipids and proteins in healthy tissues (muscle, mucosa and adipose), and 3
diseased tissues (cancerous, dysplastic, inflamed), determined using the linear
spectral decomposition approach. (Reprinted from the Ref. [343])

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on the literature data, the water / fat / protein ratio for typical soft, liquid
and hard tissues was determined in Ref. [343]. In Fig. 66 (a), we show that the
content of water in muscle, epithelial, and adipose tissue is approximately

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75, 65, and 20%, respectively [343]. Results, obtained via the spectrally-
averaged dielectric coefficient approach based on the data of THz reflection-
mode measurements, are shown in Fig. 66 (b). The reasonable data for water

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content in the different diseased and healthy tissues, compared to results in
Fig. 66 (a), were obtained; the protein content was underestimated, while the
lipid content was overestimated. Results for the linear spectral decomposition

SC
approach are shown in Fig. 66 (c). This approach revealed worse results, as
compared to the abovementioned method of the spectrally-averaged dielectric
coefficient. The water and protein content was underestimated, while the
lipid content was overestimated. However, the water concentration for the

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diseased tissue was higher compared to that of the healthy one.
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The linear spectral decomposition approach is based on determining an
absorption coefficient of pure components of tissue measured in THz trans-
mission mode. Water, lipids and gelatin represent the most commonly used
components of the tissue phantoms. In the linear spectral decomposition ap-
M
proach, the total absorption coefficient αphantom of the phantom is represented
by the weighted sum of the absorption coefficients αn of the component
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αphantom = (84)
X
α n cn ,
n
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where cn is the concentration of the nth -component; n is an integer. The

Beer-Lambert law underlies the linear spectral decomposition approach for
determining of each component concentration; it is typically used for low-
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scattering media. As follows from Eq. (84), the estimation of concentrations

can be presented in a matrix form, while the measurements of phantom
absorption coefficient should be performed at least at n wavelengths (λ1 , λ2 ,
..., λn ) for a sample comprised of n distinct absorbers.
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As a representative example of this linear model applications, we consider

a water solution of proteins, where the macromolecule is surrounded by the
AC

water hydration layer – i.e., the hydration shell with the thickness of about
1–2 nm [58]; see Fig. 24. As abovementioned in section 5, in the first approx-
imation, the absorption coefficient of the solution could be described as the
additive contribution of the solute, the solvent and the hydration shell; see
section 5, Eq. (49).
Another two approaches for measurement of the the phantom composi-

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Figure 67: THz reflection-mode images of gelatin phantoms, containing the
two round discs in the center of each image, at (a) 83, (b) 85, and (c) 87% hy-
dration. Left and right round discs are evaluated independently. (Reprinted
from the Ref. [345])

U
AN
tion that use the spectrally averaged dielectric coefficients and the Debye
relaxation model of the complex dielectric permittivity are completely de-
scribed in the thesis by Reid; see Ref. [343]. It has been demonstrated that
the THz spectroscopy is sensitive to concentration and material composition
M
of the two- and three-phase phantoms, which pure compounds were distilled
water, lipids, gelatin, and methanol.
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12.3 Phantoms imitating the degree of tissue hydra-

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tion
Since water is a primary component of biological tissue, to determine its
dielectric response at THz frequencies, it is convenient to mimic the THz-
EP

wave – tissue interactions using phantoms featuring different water concen-

tration [345]. The physiologically relevant water content in a tissue varies in
the range between 10 and 95%. Due to high water content in gelatin, the
gelatin-based phantoms has been chosen as a suitable hydration calibration
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targets for the reflection-mode THz spectroscopy and imaging. In Fig. 67,
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we show that intensity in the reflection-mode THz images of gelatin phan-

toms, each of which is comprised of the two round gelatin discs, strongly
increases with percentage of water content; as a result, it was calculated
that the hydration sensitivity of the reflection-mode THz imaging is about
0.0209–0.0389%.

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Figure 68: THz reflection-mode imaging of the IDC and fibrous tissue phan-
toms, covered by a 400-µm-thick layer of the fat tissue phantom: (a) a photo
of a sample holder; (b) a cross-section of the 3D THz-TDS data set in the
time domain and in the depth direction; (c) a cross-section of the 3D THz-

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TDS data set in the time domain and the lateral direction, at the plane
corresponding to the fat – Fibro/IDC tissue interface. (Reprinted from the
Ref. [346])
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12.4 Phantoms imitating the cancer of the breast
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The most challenging aspect in the developing tissue-equivalent phantoms
is to imitate the malignant tissues in the THz range. For example, the main
D

goal of the paper by Bowman et al. [346] was to estimate the depth, at
which the malignancy is located, and to examine of how far the normal
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tissue is located from the margins of the excised malignancy. The phantoms
were designed to mimic optical properties of the infiltrating ductal carcinoma
(IDC) – i.e., breast cancer, the fibroglandular tissues and the adipose tissues
using the commercially available mixtures of TX151, imitating the malignant
EP

and fibrous tissues, and the oil emulsions, mimicking the fat tissues. Each
phantom features the THz optical properties, which agrees well with the
literature data. In Fig. 68, we illustrate strong reflection from the IDS region,
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as compared with the reflection from the fibrous tissue phantom, even when
these tissue types are located under a 400-µ-thick layer of the fat tissues.
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12.5 Phantoms of the skin for studying the drug de-

livery
The current methods of studying the drug distribution in the skin demand
its labeling with fluorescence and radioisotopes, or applications of some mod-

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ern imaging modalities, such as the optical coherence tomography [351, 352].
In turn, the reflection-mode THz imaging could be applied for the real-time
non-invasive tracking of a chemical composition of the skin; thus, revealing

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the transdermal visualization without removal of tissues. In order to study
the capability of the THz technology in this demanding branch of drug deliv-
ery, the gelatin phantoms with an addition of the drug media (the dimethyl

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sulfoxide (DMSO) and the anti-inflammatory drug ketoprofen), featuring the
percent-by-weight of 20, 10, 5, and 2.5%, were developed [344].
It was shown that the reflection-mode THz imaging can determinine the

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content of the drug (the ketoprofen dissolved in DMSO) in the tissue phantom
(see Fig. 69 (a)), as well as in the hairless mouse skin, both ex vivo and in
vivo (see Figs. 69 (b) and (c), respectively). From Fig. 69, we can clearly
observe the round areas, representing a localization of the drug medium with

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the dissolved ketoprofen. DMSO may increase the THz reflectivity of the
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phantom and the tissue, because it is rather polar solvent. In contrast,
ketoprofen possesses a much lower absorption coefficient and refractive index
in the THz range; thus, leading to the lowering of the optical constants and
decreasing the THz reflectivity of the mixture.
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12.6 Summary
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In this section, a noticeable change in the tissue temperature was demon-

strated to be originated during the tissue exposure to THz waves with an
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average power higher than a few milliwatts. The histogram, illustrating heat-
ing of the tissue sample, being exposed to radiation of common THz sources,
was plotted. We discussed optical clearing agents (commonly referred as
THz-PEAs), that has been validated for the THz application and allows for
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decreasing the refractive index and the absorption coefficient of tissues in the
THz range, as well as increasing the difference between the THz responses
of normal and pathological tissues; thus, promising new opportunities in
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biophotonics. Finally, modern tissue phantoms for THz applications were

discussed, with a strong emphasis on the phantoms, imitating the water /
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fat / protein ratio in typical biological fluids, soft and hard tissues, mimick-
ing the THz optical properties of malignancies, or allowing for studying the
drug delivery into a tissue.

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(a)

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(b) (c)
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Figure 69: Reflection-mode THz images of (a) a tissue-equivalent phantom
with various ketoprofen concentrations and of (b),(c) a skin with various
ketoprofen concentrations ex vivo and in vivo, respectively. (Reprinted with
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permission from the Ref. [344], 2018 IEEE)

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13 Biomedical applications of high-resolution

terahertz spectroscopy
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In this section, we consider application of the high-resolution THz spec-

troscopy in analytical chemistry, biology and medicine. The high-resolution
THz spectrometers and the results of their use for studying gases and liquids
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in the listed applications are discussed. A special attention is paid to the im-
provement of the THz spectral analysis sensitivity, and to the combination
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of measurements in the THz band with that performed in the mid-IR range
using QCLs. The future trends would involve development of new miniature
and easy-to-use systems, combining radiation sources of different frequency
ranges. It would yield new analytical tools, which provide comprehensive
chemical information for both the fundamental and applied researches.
The high-resolution spectroscopy in the microwave and THz ranges is

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a powerful tool of analytical chemistry. The microwave range, spanning

the frequencies of 10 to 300 GHz, was historically the first one used in the
analytical applications. In turn, the THz frequency range includes intensive

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and sharp absorption lines corresponding to the rotational and low-frequency
vibration modes of many molecules in a gas state, which makes it attractive
for the analytical chemistry and stimulates worldwide developments in the

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high-resolution THz spectroscopy. Nowadays, the most common technique is
based on quasioptics instruments, based on the pulsed or CW PCA emitters
and detectors, or the THz wave generation and detection exploiting non-

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linear conversion of optical radiation in crystals; for example, see the review
paper [353]. The quasioptics techniques provide typical spectral resolution of
about 0.1 cm−1 (' 3 GHz), which is enough for some applications, where one
expects wide absorption bands like in semiconductor materials, liquids and

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biological tissues. Although, many problems of analytical chemistry, such
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as detection and quantification of gases in the multi-component mixtures,
require much higher frequency-domain resolution.
Methods of the high-resolution microwave and THz spectroscopy (HIRETS)
are originated mostly from microwave physics. The first microwave spectrom-
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eter exploiting the principles of FTIR spectroscopy was proposed by Flygare
in Ref. [354], where rather high sensitivity and resolution were demonstrated.
Then, this technique has been rapidly developed, improved and advanced in
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the THz range. The spectrometers employ a phenomena of the transient ab-
sorption, which arises when the radiation is brought into the resonance with
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the two level system. Thus, moving a transition into or out of resonance, one
gets alternation of macroscopic polarization’s induction and decay. The free
induction decay signal, containing information about relaxation time and
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molecular concentration, is detected and analyzed. Another phenomenon,

used in the transient experiments, is fast passage, when a frequency is swept
through a molecular resonance in a short time relative to the relaxation pro-
cess. The fast passage experiment in its present-day mode was reported in
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Ref. [355], where authors discussed reconstruction of the absorption line from
the detected signal.
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In the microwave range, for the fast passage experiments, an arbitrary

waveform generator (AWG) is used that creates a fast chirp microwave pulse
(with a linear frequency sweep and a high phase reproducibility) to polar-
ize a molecular gas [356]. The microwave synthesizer together with p-i-n
diode switches provide electromagnetic pulses for the transition absorption
(emission) experiments. Advantage of the high-resolution THz spectroscopy

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is achieved mostly by using the generators based on amplification and mul-

tiplication chains driven by microwave sources, such as AWGs and Gunn
diodes. The multiplication chains are commercially available; they yield a

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mW-power level in the sub-THz range. Another radiation source, suitable for
the high-resolution spectroscopy, is BWO generators operating mostly in the
microwave and sub-THz ranges, while the maximal frequency of the BOW

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operation reaches 1.5 THz [125]. Advantages of the BWO generators include
among others a wide frequency tunability and a high output power reaching
hundreds of milliwatts. Being voltage-controlled oscillator, the BWO gener-

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ators can be employed both for the fast passage and transition absorption
(emission) experiments.
Another approach in the HIRETS development involves difference-frequency
generation in PCA using two CW lasers [357]. This technique provides radi-

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ation up to 2.6 THz, as well as the broadband frequency tunability and the
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high frequency-domain resolution of several MHz.
In Fig. 70, we show a schematic of the HIRETS system. In this scheme, a
radiation source is phase (frequency) stabilized by the phase lock loop system
(PLL) and provided by the phase modulation or the frequency sweeping sys-
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tems, depending on the experiment type. The response of the gas molecules
is detected by the receiver and further analyzed. Among components of
the spectrometers are, in particular, the devices for upward and downward
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frequency conversion (multipliers, harmonic mixers), as well as the signal

generators. A CW THz radiation with frequency stabilization is provided by
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a BWO-based synthesizer, for which the generation bandwidth is less than

10 Hz.
The promising research direction in the area of analytic spectroscopy of
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gases is associated with taking into account those frequency ranges, where
gases possess maximal absorption. For example, the multichannel spectrom-
eter combining two spectral ranges was developed in Ref. [358]. The spec-
troscopic methods make it possible to work with any aggregate state (solid,
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liquid, or gas) in the THz range, where we can observe low-frequency molec-
ular vibrations, in which large groups of atoms in a molecule move as a unit
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(e.g., nucleotide in DNA). The frequency of these oscillations depends on the

structure of the whole molecule. Furthermore, oscillation frequencies of the
hydrogen bonds, supporting the structure of biomolecules, are also located
in the THz band. The lines of water absorption are also located in the THz
range; which are dependent on vibrational-rotational transitions and relax-
ational dielectric losses. Water absorption bands allow us to estimate the

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amount of water in a sample.

Thus, modern technical improvements have significantly expanded the
scope of high resolution spectroscopy from the small gaseous molecules to the

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larger systems with multiple degrees of freedom and even to the substances in
liquid or solid states. The precise information about the central frequencies,
the width and profiles of the absorption lines, provided by the spectrometers,

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is used for calculation of the rotational constants and the absorption lines
broadening. Then, the detailed information about molecular spectra is used
for solving various problems in analytical chemistry.

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The main model applied for describing the rotational spectra includes
rigid rotor approximation – i.e., a molecule rotates as a rigid body. In this
case, the spectra are determined by rotational constants, which are inverse
proportional to the moments of inertia. To accurately account for the posi-

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Figure 70: A scheme of the high-resolution THz spectrometer; here, BWO

operates at 118–178 GHz; Att. stands for the attenuator, ADC1,ADC2 stand
for the analog-to-digital converters. (Courtesy of V.L. Vaks)

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tions of the rotational transitions, the model is supplemented by the ”non-

rigidity” effects – centrifugal stretching and vibration-rotation coupling. The
spectra can be further complicated by the coupling between the electronic

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motions and the molecular rotation. Moreover, the rotational spectra can
have the nuclear hyperfine structure, arising from either magnetic or elec-
tric interactions of the molecular fields, generated by electrons, with the

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nuclear spins, or from a combination of these two factors [359]. The nu-
clear quadrupole hyperfine structure provides a measure of the molecular
field gradients, from which much information about the electronic structure

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and chemical bonds can be obtained. The precision of the up-to-date THz
spectrometers is enough to observe all the listed effects.
Apart from central frequency and intensity, an absorption lines width is
often required for analytical applications. First of all, the lines width is used

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to calculate a concentration of a gas under study [360]. The percentage of

Nν =
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gas can be derived from a simple proportion
γexp ∆νexp
× 100%, (85)
γtheor ∆νtheor
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where γtheor and ∆νtheor are the absorption coefficient and linewidth for the
100%-concentration correspondingly; while γexp and ∆νexp represent similar
parameters observed in the experiment.
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The high resolution of this type of THz spectroscopy allows for discrim-
inating of all various conformers or isotopic species. Determination of the
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latter by their rotational frequencies benefits from the

q fact that the ratio
of isotopic rotational frequencies is m1 /m2 instead of m1 /m2 for vibration
frequencies, where m1 and m2 are masses of the two isotopes. Studies of
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rotational spectra of water isotopologues (HD17 O and D17 2 O) in the range

0.65–1.6 THz have provided rotational, centrifugal-distortion constants and
the oxygen quadrupole-coupling constants [361]. The hyperfine structure de-
pends strongly on the electronic environment of the amino nitrogen atom
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and its orientation towards the principal inertial axis. The approach was
employed for the identification and studying conformers of tryptamine [362].
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The quadrupole hyperfine structure in the spectra of the two conformers of

tryptamine, as an example, is shown in Fig. 71 [362].
The molecular complexes are aggregates of a few molecules (or molecules
and atoms), which are bound very weakly. The complexes are produced by
cooling the gas to cryogenic temperature during supersonic jet expansion. An
extensive review on studies of molecular complexes by microwave and THz

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spectroscopy is presented in Ref. [363]. An analysis of the rotational spectra,

followed by the calculation of rotational constants, reveals the structure of
complexes.

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Studies of biological and biologically active molecules in the gas phase
provide information about their native structure, which is not influenced by
the environment; thus, revealing the intramolecular factors controlling the

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molecular properties and the structure-function relationships.
One of the effective methods of non-invasive medical diagnostics is anal-
ysis of a content of the exhaled breath and the ”odors” of biological liquids

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and tissues. It can be used for determining the diseases and for predicting
the organism reactions in case of the specific treatment. The oncological,
inflammatory, endocrine and other diseases can be diagnosed by measuring
the concentration of markers in exhaled breath. The spectroscopic meth-

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ods, including spectroscopy in the THz range, show considerable promise
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among other methods of exhaled breath analysis. The results of spectro-
scopic investigations of exhaled breath for non-invasive medical diagnostics
of diabetes, precancerous conditions and oncological diseases of gastrointesti-
nal tract organs, as well as for monitoring the effectiveness of radiotherapy
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by dynamics of nitric oxide concentration, highlight a bright prospective of
the high-resolution THz spectroscopy in medical diagnosis [364].
One of the important applications of THz spectroscopy is associated with
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Figure 71: The quadrupole hyperfine structures of the tryptamine conform-

ers, due to the presence of two 14 N nuclei. (Reprinted from the Ref. [362])

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the development of novel methods for non-invasive detection of the confor-

mational state of biological molecules in fluids or dry samples. Functions of
biomolecules, such as DNA and proteins, depend not only on their chemical

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composition, but also on the spatial structure of the molecules – i.e., so-called,
configurational and conformational state. The conformational transitions of
the biomolecules play an important role in the processes, taking place both

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in healthy and pathological cells, e.g., diseases such as Alzheimer’s disease,
Huntington’s, Parkinson’s, or even type 2 diabetes, can be attributed to con-
formational diseases, i.e., diseases caused by the formation of proteins with

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abnormal conformation.
A significant problem for the study of biomolecules in an environment
close to natural is the strong absorption of THz frequency radiation by liquid
water. Therefore, in many studies the samples were specially prepared, for

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example, cryogenically frozen, dried, pressed into tablets or pellets, etc.
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In summary, nowadays the HIRETS is mostly used in fundamental re-
search in molecular physics. Although, together with an improvement of the
sensitivity, this technique can replace other powerful analytical techniques
like gas-chromatography and mass spectrometry, when it comes to trace gas
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detection and quantification. We believe that progress in semiconductor
technology will result in a very good level of performance of sources and de-
tectors for THz spectroscopy. Future trends would involve the development
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of new miniature and easy-to-use instruments, combining radiation sources

of different frequency ranges. It would yield new analytical tools to provide
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better chemical information both for fundamental and applied studies. The
promising candidates for the IR radiation sources are QCL operating in the
mid-IR range. These are the small devices, spanning the wavelengths range
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from 4.0 to 24 µm and providing pulsed or CW radiation with the output

power up to 3 W at an ambient temperature.
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Conclusions
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Water is the basis of life on Earth and the study of its role in life takes a
dominant place in the work of researchers studying biological systems. The
present paper is an attempt to identify the main trends in the use of THz
radiation in biological and medical research where water is the primary object
of study. It is known that THz spectroscopy methods are sensitive not only
to water, but to other specific structural and molecular features of biological

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systems. However, in this work we are focusing mostly on water which can
take various forms in biological molecular structures and tissues. It can also
play the key role in the formation and stabilization of biosystems and is the

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universal marker of their state and their functioning. Being well aware that
it is the dielectric properties of water in different states that form the basis
for diagnostic methods, we started our review with the analysis of physical

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models describing a complex dielectric permittivity of liquid water and water-
containing media in the THz frequency range. However, this analysis also
affects the neighboring/next electromagnetic frequency ranges. The models

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described in the review reveal relaxation and damped-resonant picosecond
dynamics underlying the THz dielectric response of such media. They yield
analytical and numerical modeling of THz wave interaction with water and
water-containing matter, such as biological tissues and fluids. The goal of

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this review is to draw attention of the Quantum Electronics community to
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the importance of the development of dielectric models of water-containing
media in the THz frequency range.
Then, we briefly discussed modern instruments and methods of THz spec-
troscopy and imaging, which are widely applied in THz biophotonics. Among
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them, we pay a special attention to the THz time-domain spectroscopy and
related imaging modalities, which remain the dominant research instruments
used in THz science and technology due to their simplicity, technical reliabil-
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ity and high information content of the measured data. We also considered
modern developments in the area of sub-wavelength-resolution THz imaging,
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which attracts considerable attention in fundamental and applied physics

and, obviously, their applications in biology and medicine, allowing for visu-
alizing such structural components of tissues as cells, microfibrils, and even
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cell organelles. The problem of THz wave delivery to/from tissues that are
hard to access and internal organs still remains challenging, restricting ap-
plications of THz diagnosis. These difficulties are associated with the lack of
the hard waveguides or flexible fibers capable for operation at THz frequen-
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cies. Were have reviewed recent developments on THz waveguides relying on

various physical principles, exploiting novel materials and modern fabrica-
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tion technologies. We summarized the main tendencies in this research field

and highlighted the most promising ones.
Although the strong absorption of THz-waves by water molecules prevents
them from penetrating hydrated tissues and analyzing the THz response of
other tissue components, we discuss novel approaches for overcoming this
drawback relying on the freezing of tissues ex vivo or the immersion optical

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clearing of tissues ex vivo or in vivo. These beneficial techniques have a bright

future in THz biophotonics, particularly in cancer detection. One of the most
attractive biomedical applications of THz spectroscopy and imaging is associ-

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ated with the label-free diagnosis of malignancies. Therefore, in this review,
we discuss recent research results in this area, considering malignancies in
different localizations and classified methods as for noninvasive, least-invasive

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and intraoperative diagnosis, and THz histology. We paid attention to the
origin of contrast observed between healthy and malignant tissues in the
THz range, and we discussed difficulties of the transfer of THz technology

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into the clinical environment. We think that further developments in this
attractive field of THz biotechnology is associated with both accumulation
and analysis of verified database of THz dielectric responses of healthy and
malignant tissues, in-depth study of the nature of label-free contrast and,

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finally, development of portable, low-cost, and ergonomic instruments of the
THz diagnosis. AN
Besides the THz diagnosis of malignancies, we considered applications of
THz reflectometry in sensing the thinning dynamics in a human precorneal
tear film. The results of these studies show a potential of THz technology
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in ophthalmology. We described modern multi-spectral and multi-temporal
THz imaging modalities, realizing the principles of color vision, phase analy-
sis and tomography, as well as modern approaches of the THz spectra anal-
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ysis, based on machine learning, pattern recognition and chemical imaging.

They reveal spatial distribution of various substances in a tissue and might
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be useful in THz biomedicine.

We discussed a new thermal model describing the biological object irra-
diated by THz radiation, as well as existing agents allowing for enhancing
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the THz-wave penetration into tissue by its temporal and reversible dehy-
dration, and phantoms mimicking the optical properties of tissues at THz
frequencies. Thereby, this review paper has allowed us to discuss the rapid
progress in the THz biophotonics, highlight modern research and engineer-
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ing problems in this exciting field of THz technologies, and demonstrate its
bright prospectives.
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Acknowledgements
We are grateful to all our colleagues who, not being co-authors, nev-
ertheless have made valuable contributions to the article content and our

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understanding of future of THz biophotonics. It is hard to overestimate the

support of Prof Martina Havenith, Dr. Peter H. Siegel, Dr. Gian Piero
Gallerano and Dr. Andrea Markelz at all stages of the work on this review

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and the useful discussions we had.
The reported study was funded by the Russian Foundation for Basic
Research (RFBR) according to the Cooperation Research Project KOMFI,

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Project # 17-00-00275 (# 17-00-00270, # 17-00-00272, # 17-00-00184, # 17-
00-00186).
This work was also partially supported by Basic Research Program of

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the Presidium of the Russian Academy of Sciences # 32; the RFBR grants
# 15-29-03900, # 16-52-76011 ERA_a, # 16-29-11763, # 17-02-00358, # 17-
08-00803, # 17-38-80057, # 18-52-16025, # 18-38-00504, and # 18-29-02060;
Lomonosov Moscow State University Program of Development, the Council

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of RF President grant # NSh-9695.2016.2; the RF MES grant for the In-
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crease Competitiveness Program of NUST ”MISiS” (# K2-2017-003), Rus-
sian academic excellence project ”5-100” for Sechenov University, and grant
# 17.1223.2017/AP; the RF Government grant # 08-08, and the Technolog-
ical Platform ”The Medicine of Future”.
M
The work of N.V. Chernomyrdin and K.I. Zaytsev on sections 4.3 and
6 was supported by the Russian Science Foundation (RSF), grant # 17-79-
20346; O.P. Cherkasova on section 5 - the RSF grant # 18-12-00328; and
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V.L. Vaks on section 13 – the RSF grant # 15-12-10035.

The work on section 5.7 was supported by the National Research Foun-
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dation of Korea (NRF), grant funded by the Korean Government (MEST)

(# 2017R1A2B2007827), and by Institute for Information & Communica-
tions Technology Promotion (IITP), grant funded by the Korea Government
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(MSIP) (# 2017-0-00422).
The work of Patrick Moneaux on section 13 was supported by New Aqui-
tania Region for Farsense Project.
English editing of the manuscript was done by Vincent Wallace.
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