LA SER S • OPT IC S • IMAGIN G • S P E C TROS CO PY • MICR OS COPY September 2019

VIS-OCT Brings
Vascular Network into Focus

www.photonics.com

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919_NKTPhotonics_Pg03.indd 3 9/5/2019 8:22:47 AM
 LA S ER S • OPTICS • IMAGI N G • SP E C T R O S CO PY • MICR O SCO PY Volume 26 • Issue 6

® www.photonics.com

NEWS
10 • BIOSCAN
BioPhotonics editors curate the most significant headlines for photonics
in the life sciences — and take you deeper inside the news.
Featured stories include:
• Wearable NIRS device reveals how seals prepare to dive
• Two-photon microscope captures brain activity at record speed
• Terahertz imaging system on a chip offers speed and portability

17 • RAPIDSCAN
10 • Prellis Biologics receives $8.7M, reports tissue printing progress
• Olympus invites entries for Global Light Microscopy Image of the Year

FEATURES
22 • VIS-OCT OPENS EYES TO NEW MEDICAL APPROACHES
by Hao F. Zhang, Northwestern University, and Kieren Patel, Opticent Health
Attention and investment could revolutionize applications in visioning
and treatment of eye-related diseases.

28 • LENS-FREE MICROSCOPY FOCUSES ON COMMERCIALIZATION

by Hank Hogan, Contributing Editor
Years after Aydogan Ozcan’s demonstration, the technology evolves to
meet real-world applications.

32 • FLUORESCENCE MICROSCOPY: GETTING THE PICTURE RIGHT

by Felix Asche, Basler AG
Camera choice has to strike a balance between certain sensor properties,
32 camera-related topics, and the needs of the intended applications
in industry or medicine.

38 • OPEN-SOURCE PHOTON COUNTING

THE COVER
VIS-OCT delivers images
of retinal layers of a mouse
with high resolution and
accuracy. See page 22.
Courtesy of S. Chen et al.,
Biomed Opt Exp. Cover
design by Art Director
Suzanne L. Schmidt.
ADVANCES BIOLOGICAL RESEARCH
by Pablo Blinder, Lior Golgher, and Hagai Har-Gil, Tel Aviv University
Fast and volumetric intravital imaging represents the most demanding scenario
for which photon counting is drastically improving the performance of existing
systems.
42 • NEUROSCIENCE 2019 CELEBRATES SFN’S 50TH ANNIVERSARY
The world’s largest neuroscience conference focused on the brain and nervous
system marks the 50th anniversary of the Society for Neuroscience (SfN).
44 • Q&A WITH IWAN W. SCHIE
Spectroscopy gives clear view of data

COLUMNS AND DEPARTMENTS

PHOTONICS 6 • EDITORIAL
The technology of generating and harnessing light
and other forms of radiant energy whose quantum unit 7 • BIOPINION
is the photon. The range of applications of photonics Illumination in microscopy often overlooked
extends from energy generation to detection to
communications and information processing. 46 • BREAKTHROUGH PRODUCTS
BIOPHOTONICS 48 • APPOINTMENTS
The application of photonic products and techniques Upcoming courses and shows
to solve problems for researchers, product developers,
clinical users, physicians, and others in the fields of 49 • ADVERTISER INDEX
medicine, biology, and biotechnology.
50 • POSTSCRIPTS
The faster the flow, the brighter the glow

4 BioPhotonics • September 2019

919BI_Contents.indd 4 9/5/2019 8:10:02 AM

 LASERS • OPTICS • IMAG ING • SPEC TROS CO PY • MICR OSCOPY

®
www.photonics.com
BioPhotonics Editor Douglas J. Farmer

Editorial Staff
Editor-in-Chief Michael D. Wheeler
Senior Editor Susan M. Petrie
Senior Editor Douglas J. Farmer
Associate Editor Joel P. Williams
Multimedia/Web Editor Robin J. Riley
Chief Copy Editor Carol A. McKenna
Copy Editors Cheryl L. Hulseapple, Bill D. Latimer
Contributing Editors Hank Hogan, Farooq Ahmed,
Marie Freebody

Creative Staff
Senior Art Director Lisa N. Comstock
BioPhotonics Art Director Suzanne L. Schmidt
Creative Designer Devon A. Unwin
Designer Dina J. Oliveira NEW
Digital Media & IT Staff
Director of Publishing Operations Kathleen A. Alibozek CELESTA
7-LINE LASER LIGHT ENGINE
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Digital Developer & IT Support Brian A. Bilodeau IT JUST KEEPS GETTING
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BioPhotonics • September 2019 5

919BI_Masthead.indd 5 9/5/2019 8:32:20 AM


BioPhotonics Editorial Advisory Board
Mark A. Anastasio, Ph.D.
Professor of Biomedical Engineering
Washington University in St. Louis
David Benaron, M.D.
Professor, Medicine (consulting)
Founder, Stanford Biomedical Optics program
Stanford University School of Medicine
CEO, Spectros Corp.
Stephen A. Boppart, M.D., Ph.D.
Bliss Professor of Engineering
Electrical and Computer
Engineering, Bioengineering and Medicine
Beckman Institute for Advanced
Science and Technology
University of Illinois at Urbana-Champaign
Aydogan Ozcan, Ph.D.
Chancellor’s Professor, Electrical & Computer
Engineering Department
University of California, Los Angeles
HHMI Professor, The Howard Hughes
Medical Institute
Adam Wax, Ph.D.
Professor of Biomedical Engineering
Duke University
Founder and President, Lumedica
Submit your press releases to pr@photonics.com,
6 or use our online submission form at www.photonics.com/prsubmit.
919BI_Editorial.indd 6
EDITORIAL
VIS-OCT offers sight line
T
he benefits of optical coherence tomography (OCT) have long been known as an
imaging tool in ophthalmology, enabling the examination of the eye at microscopic
resolution. But what may not be widely appreciated is that the use of visible light
sources can improve upon the results of this well-developed technique in the detection
and understanding of various eye-related diseases such as macular degeneration and
glaucoma.
While most commercial OCT and OCTA (angiography) systems use NIR light for
economic and technical reasons, broadband visible light sources have expanded from 400
to 2000 nm or more. This has enabled the imaging of the complete vascular network and
all five of the outer retinal layers. With resulting higher resolution and imaging contrasts
at the tissue level generated with VIS-OCT made available to clinicians and researchers,
conditions like blood oxygen saturation can be measured as never before.
In our cover story, professor Hao F. Zhang of Northwestern University and Kieren
Patel, a founder of Opticent Health, reflect on the many possibilities for ophthalmology
brought to the forefront with VIS-OCT. More investment in VIS-OCT is needed, they
write on page 22, to bring down costs and excite clinicians about its application.
Also featured in this issue:
• In lens-free microscopy, a light source is placed right on top of an image sensor. An
algorithm helps translate the pattern caught by the sensor to achieve enough resolution
that the image can be studied and virtually dissected. Contributing editor Hank Hogan
talks to experts in the field, who reveal that challenges remain to fully implementing
lens-free microscopy in the laboratory and field. Without the need for lenses, the micro-
scopes can be made smaller and less expensive than other types. Read more on page 28.
• Keeping with the core technologies of microscopy, guest authors Pablo Blinder, Lior
Golgher, and Hagai Har-Gil of Tel Aviv University write that open-source photon count-
ing can reveal details of a specimen while removing much of the noise. PySight, an open-
source application, has been developed to implement this technology in standard micro-
scopes, eliminating the need for extensive training or upgrades. Using a depth-scanning
varifocal lens, researchers were able to acquire a large volume of images, capturing what
is occurring in a specimen in real time. Read more on page 38.
• When searching for a suitable camera to take advantage of the dynamic developments
within the spectrum of fluorescence microscopy, prospective buyers should consider a
number of factors, Felix Asche of Basler points out. Basic choices — including optical
format and resolution; CCD, CMOS, sCMOS, or BSI; monochrome or color; global
shutter versus rolling shutter; sensitivity and dynamic range; image quality and noise;
and cooling — all weigh on the search to acquire a camera for purposes in a range of
industrial settings. Asche explains that with SONY’s discontinuation of CCD camera
components, opportunities exist for companies to chart new ground in the life sciences.
Get an inside look at some of these changes on page 32.
• And in our “Biopinion” for this issue, Jasmin Schaefer, Iain Johnson, and Claudia
Jaffe of Lumencor Inc. examine the need for strong, high-quality light sources for mi-
croscopy, a feature they argue is too often overlooked in medical and industrial applica-
tions. Solid-state lighting has been available for the last half-century, they point out,
but there are many medical and manufacturing settings where, for whatever reason,
it has not been embraced. With the right light source, test results can prove to be more
reliable and less expensive at the same time. See page 7 to learn more about the authors’
perspectives.
Enjoy the issue!
doug.farmer@photonics.com
BioPhotonics • September
BioPhotonics • April 2019
2018
9/13/2019 10:55:48 AM
 BIOPINION Views on how to advance biophotonics
Illumination in microscopy often overlooked
BY JASMIN SCHAEFER, IAIN JOHNSON, AND CLAUDIA JAFFE, LUMENCOR INC.
A
s scientists engage in the development and manufacture
of high-performance lighting, we find it frustrating to en-
counter a lack of appreciation for the illumination source
in the successful application of light microscopy. Like that other
pervasive illumination source, the sun, microscope light sources
are commonly taken for granted. It often seems they only draw
attention when they are absent. This is unfortunate, as it not only
leaves many users unaware of the benefits of modern solid-state
illumination technologies, but it leaves them reliant on archaic
incandescent bulbs and mercury arc lamps.
We cannot help but observe how stark the contrast is with the
technologies on the detection side of the microscope. During
the last 30 years, emulsion film recording has been completely
displaced by solid-state digital cameras. While solid-state light-
ing has been around for more than 50 years, we have yet to see
its full appreciation or similarly broad adoption in academic
spheres, biotech laboratories, hospitals, and clinical labs, or by
biotech equipment manufacturers.
The benefits of solid-state light sources are significant.
Their nonreliance on mercury and other hazardous substances
and their minimal operation and maintenance costs are well
documented. A typical HBO100W bulb used in fluorescence
microscopy contains about 20 mg of mercury and has a surface
temperature of 800 °C and an internal pressure of 30 to 75 bar
(400 to 1100 psi) under typical operating conditions. Under such
challenging conditions, operational lifetime is only 200 to 300
hours. In stark contrast, LEDs used in lighting systems with ad-
equate heat dissipation operate at ambient temperature and pres-
sure and are capable of more than 10,000 hours of user operation
(equivalent to 5 years of 40-hour weeks). Solid-state illuminators
also provide superior light output stability with respect to wave-
length and power, and on both a frame-to-frame and day-to-day
basis. This is of value for quantitative comparisons where im-
ages are acquired at various intervals in a time-lapse sequence.
Solid-state light sources facilitate application-specific custom-
ization of illumination. The traditional “top-down” illumination
approach starts with a spectral distribution that is physically
invariant (e.g., the atomic emission of mercury vapor). Such
output spectra are adapted to application requirements by selec-
tive blocking and attenuation of the superfluous wavelengths and
optical power. Yet the more modern approach assembles discrete
solid-state light sources such as LEDs and laser diodes within
an electronic control framework. Such contemporary designs
enable the user by optimally matching application requirements
in terms of the spectral distribution, angular distribution, and
radiant flux (power).
The electronic control systems of today’s solid-state illumina-
tors are increasingly sophisticated. They provide not only the
basic output control functions (color band selection, output on/
off, and intensity adjustment) but also real-time performance
monitoring and feedback control. These control systems can now
incorporate Ethernet connectivity, allowing performance data to
be accessed remotely for diagnostic purposes. This will facilitate
improved troubleshooting, providing a benefit to users and
BioPhotonics
BioPhotonics •
• May/June
September 2018
2019
919BI_BIopinion.indd 7
service providers alike. We commonly observe that users of fluo-
rescence microscopes evaluate the performance of a light source
based on the camera exposure time required to acquire an image
of a fluorescent specimen. Yet this is a fundamentally unsound
practice, because many factors determine the exposure time
and only some of them directly relate to the output of the light
source. Onboard monitoring systems allow the performance of
the light source to be evaluated directly and independently of the
microscope, camera, and specimen.
In conclusion, it is our opinion that users of fluorescence
microscopy and other optical imaging techniques should keep
in mind that although the light source may be hidden in plain
sight, overlooking its importance may result in missed oppor-
tunities. The potential exists today for improved data quality,
better operational reliability and throughput, cleaner techniques,
cost-effective operation, and illumination that is customized
to the requirements of the application. To fully employ today’s
solid-state illumination, users need to pay mindful attention to
the uniqueness these state-of-the-art products offer versus their
all-too-familiar and pervasive bulb-containing predecessors.
Meet the authors
Jasmin Schaefer, technical support specialist at Lumencor Inc., earned
her Master of Science degree in biomedical engineering from Oregon
Health and Science University in Portland, Ore. There she developed
a fluorescence imaging method for rapid intraoperative tumor margin
assessment. At Lumencor, she works under Iain Johnson to aid research-
ers and internal sales staff with technical support. Her background in
fluorescence imaging has been an asset when it comes to understanding
customer applications and while troubleshooting issues that may crop up
with Lumencor light engines.
Iain Johnson, Ph.D., director of technical support
at Lumencor, is a biochemist with expertise
in the biophysics of fluorescence microscopy.
With numerous patents and publications to his
credit, he is steeped in the physical chemistry of
fluorescent and luminescent probes. For many
years he has been a faculty member at the annual
University of British Columbia 3D Microscopy
course as well as with Molecular Probes/Invit-
rogen/Life Technologies and his own consulting
firm. He is responsible for technical support of both internal sales staff
and cutting-edge researchers around the world.
Claudia Jaffe, Ph.D., is co-founder and execu-
tive vice president at Lumencor. She earned her
doctorate in bioanalytical chemistry from the
University of Pittsburgh. She has developed,
published, and patented a variety of electro-
chemical and photoelectrochemical sensors and
bioanalytical chips, focusing her efforts on high
throughput analyses employing enzymology,
immunology, and genomics. At Lumencor, she is
an inventor in nearly all of the company’s patents
and the leader of new business development. She supervises sales and
marketing as well.
7
7
9/5/2019 7:55:19 AM
 CONTRIBUTORS
Felix Asche, Ph.D., is product
market manager for the Basler
AG MED ace camera series.
Welcome to
After studying biology, he
completed his doctorate at the
Bernhard Nocht Institute for
Tropical Medicine. Page 32.
Pablo Blinder, Ph.D., leads a The online companion to BioPhotonics magazine
multidisciplinary laboratory in
the School of Neurobiology,
Biochemistry & Biophysics as
a member of the life sciences
What’s Online: Free We
faculty at Tel Aviv University in
Israel. Page 38.
binars
Lior Golgher is a doctoral Register today for these free webinars
student at the Sagol School
of Neuroscience at Tel Aviv Solving Challenges in Defect Inspection of Advanced Optics
University. Page 38. Mon, Sept. 23, 1 p.m. EDT
Presenter Samuel Lesko will explain how customized analysis can easily spot the most
minute defects, even in cases of complex surface geometry, and how it automatically
outputs critical information on detected defects, without operator intervention or an
Hagai Har-Gil is a graduate
student at Tel Aviv University.
etching step. Presented by Bruker.
He has a bachelor’s degree in To register, visit www.photonics.com/w194.
physics. Page 38.

Everything You Ever Wanted to Know About Optical Coatings

but Were Afraid to Ask
Hank Hogan, contributing edi- Thu, Sept. 26, 1 p.m. EDT
tor, has a bachelor’s degree
This interactive webinar led by the technical experts at North American Coating
in physics from the University
of Texas at Austin. He worked
Laboratories will explore the rapidly evolving field of optical coatings
in the semiconductor industry and address questions and concerns with detailed technical responses.
and now writes about science. Presented by North American Coating Laboratories.
Page 28.
To register, visit www.photonics.com/w195.
Claudia Jaffe, Ph.D., is co-
founder and executive vice
president at Lumencor Inc. OCT and Ophthalmology in the Age of Artificial Intelligence
She earned her doctorate in Tue, Oct. 8, 1 p.m. EDT
bioanalytical chemistry from Presenter Nishant Mohan provides an executive overview for leaders in imaging and
the University of Pittsburgh.
ophthalmology on how AI is transforming the field of medical imaging. Mohan will
Page 7.
demonstrate how to develop a deep learning AI system from scratch, giving attendees
Iain Johnson, Ph.D., director of
critical insight into how to use this powerful tool.
technical support at Lumencor,
is a biochemist with expertise To register, visit www.photonics.com/w193.
in the biophysics of fluores-
cence microscopy. Page 7.
High-Power Diode Laser Solutions for Manufacturing
and Scientific Applications
Kieren Patel (Ph.D., MBA, Wed, Oct. 9, 1 p.m. EDT
J.D.) is CEO of Opticent Health, Jörg Neukum from Coherent will discuss several key applications and how high-power
which seeks to commercialize
diode laser solutions are optimized for each application. Learn how innovations rang-
visible-light optical coherence
tomography for both clinical and ing from corrosion-resistant cooling schemes to novel beam shaping optics enable
preclinical markets. Page 22. diode laser manufacturers to continue to improve solutions that service these applica-
tions. Sponsored by RPMC Lasers Inc., TOPTICA Photonics, and SemiNex Corporation.
Jasmin Schaefer, technical To register, visit www.photonics.com/w188.
support specialist at Lumencor
Inc., earned her Master of
Science degree in biomedi-
cal engineering from Oregon
Health and Science University
in Portland, Ore. Page 7.
Hao F. Zhang, Ph.D., is profes- Follow Photonics Media on Facebook, Twitter, Instagram, LinkedIn, and YouTube.
sor of biomedical engineering
and ophthalmology at North-
western University. Page 22.
www.photonics.com.

8 BioPhotonics • September 2019

919BI_OnlineContribs.indd 8 9/5/2019 9:33:07 AM

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 BIOSCAN
A closer look at the most significant biophotonics research and technology headlines

Wearable NIRS device reveals how seals prepare to dive

ST. ANDREWS, Scotland — Scientists at With the PortaSeal, the scientists were submersion. These anticipatory adjust-
the University of St. Andrews have cre- able to obtain detailed continuous NIRS ments suggest blood redistribution in
ated a wearable, noninvasive device based data from four seals swimming freely in a seals is under some degree of cognitive
on near-infrared spectroscopy (NIRS) that quasi-natural foraging habitat. The device control and is not just a reflex response
can be used to investigate blood volume was superglued to the animals’ heads to submersion. “Getting this insight with
and oxygenation patterns in freely diving or shoulders and then easily removed to noninvasive wearable technology from
marine mammals, such as seals. download the data. the biomedical field offers many exciting
When mammals are submerged in The results of the study were intrigu- future research avenues,” said research
water, they show a suite of cardiovascular ing, said the researchers: Seals routinely fellow J. Chris McKnight.
responses, such as reduced heart rate and constrict their peripheral blood vessels, The research was published in PLOS
constriction of peripheral blood vessels. accompanied by increased cerebral blood Biology (https://doi.org/10.1371/journal
The researchers hypothesized NIRS volume, approximately 15 seconds before .pbio.3000306).
could provide high-resolution, relative
measures of oxygenated and deoxygen-
ated hemoglobin within specific tissues,
which then could be used to estimate
changes in blood volume. They adapted
NIRS technology for use on freely diving
harbor seals to investigate blood volume
and oxygenation patterns specifically in
the brain and blubber, using a device they
called the PortaSeal.

Visualization illustrating the underlying concept of a three-optode-receiver-channel spatially resolved

A juvenile harbor seal. Courtesy of Monica Arso
Civil/Sea Mammal Research Unit, University of
St. Andrews.
continuous-wave NIRS sensor. Heterodyning dual-wavelength light, visualized in red, is emitted from three
light-emitting optodes in contact with a seal’s skin (green arrow). Light passes through the underlying
tissue before exiting the head, where it is detected by a photodiode in contact with the seal’s skin
(red arrow). Increased distance between the optode and receiver channels provides deeper optical
penetration within the underlying tissue. Courtesy of J. Chris McKnight et al./PLOS Biology.

Two-photon microscope captures brain activity at record speed

ASHBURN, Va. — A new two-photon
microscope from scientists at Howard
Hughes Medical Institute’s Janelia
Research Campus can record footage
of brain activity 15× faster than once
believed possible, the team said, reveal-
ing voltage changes and neurotransmitter
release over large areas and monitoring
hundreds of synapses simultaneously.
The new tool, called scanned line
angular projection microscopy, or SLAP,
makes data collection more efficient by
compressing multiple pixels into one mea-
surement and scanning only the pixels
in the areas of interest. A device controls
which parts of the image are illuminated,
and thus which parts are scanned. A high-
resolution picture of the sample, captured
before the two-photon imaging begins,
guides the scope and allows scientists to
decompress the data to create detailed
videos.
Much like a computed tomography
scanner, which builds up an image by
scanning a patient from various angles,
SLAP sweeps a beam of light across
a sample along four different planes.
Instead of recording each pixel in the
beam’s path as an individual data point,
the scope compresses the points in that
line together into one number. Then,
computer programs unscramble the lines
of pixels to get data for every point in
the sample. SLAP can computationally
recover high-resolution images attaining
voxel rates of over 1 billion Hz in struc-
tured samples.
In classic two-photon microscopy, each
measurement takes a few nanoseconds.
Making a video requires taking mea-
surements for every pixel in the image
in every frame. This, in theory, should
limit how fast one can capture an image,
research fellow Kaspar Podgorski said.
However, the new microscope from

Submit your press releases to pr@photonics.com,

10 or use our online submission form at www.photonics.com/prsubmit. BioPhotonics • September 2019

919BI_BioScan.indd 10 9/5/2019 7:56:43 AM

 Podgorski’s team surpasses these limits, An artist’s impression of how the HHMI team
achieving a speed that could previously labeled individual neurons in a mouse’s brain with
a fluorescent protein that makes synapses flash
be achieved only over tiny areas.
when activated (red stars). The SLAP micro-
In the time it takes SLAP to scan the scope records those flashes along four different
whole sample, a traditional scope going axes (red lines). Then a computer program
pixel by pixel would cover just a small reconstructs which synapses were active at any
fraction of an image. This speed allowed given time based on how the signals on the four
axes overlap. Courtesy of Ella Marushchenko.
Podgorski’s team to watch in detail how
glutamate, a neurotransmitter, is released
onto different parts of mouse neurons.
The team’s ultimate goal is to image brains, that are impenetrable with regular
all of the signals coming into a single light microscopy. Until the introduc-
neuron, so they can better understand how tion of SLAP, it had not been possible to
neurons transform incoming signals into capture the patterns of neurotransmitter
outgoing signals. This current scope is release onto neurons in the brains of liv-
“only a step along the way — but we’re ing animals at a millisecond timescale,
already building a second generation,” according to the team.
Podgorski said. The research was published in Nature
Scientists use two-photon imaging to Materials (https://doi.org/10.1038/s41592-
look inside opaque samples, such as living 019-0493-9).

Terahertz imaging system on a chip offers speed and portability

PRINCETON, N.J. — An imaging system histidine. An 81- × 53-pixel hyperspectral can occur when the active ingredient is not
developed at Princeton University uses image was acquired through a raster scan properly mixed into a tablet.
lasers small enough to fit on a microchip, of a solid pressed disk containing the three To demonstrate the quality of the im-
and emits and detects electromagnetic differently absorbing compounds. The age resolution, the team imaged a U.S.
radiation at terahertz (THz) frequencies. THz imaging system identified each ingre- quarter. Details on the coin as fine as the
The new system, which is based on a dient and revealed the boundaries between eagle’s wing feathers and as small as one-
semiconductor design, uses a dual-comb them, as well as a few spots where one fifth of a millimeter were clearly visible.
structure to efficiently measure the radia- chemical had spilled over into a different Although this system makes the in-
tion that is reflected from the sample. zone. This type of “hot spot” represents a dustrial and medical use of THz imaging
Spectral signatures in the reflected radia- problem in pharmaceutical production that more feasible than before, it still requires
tion enable researchers to identify the
sample’s molecular makeup.
The device performs hyperspectral
imaging with chip-scale frequency combs
based on THz quantum cascade lasers.
The dual combs are free-running and
emit coherent THz radiation that covers
a bandwidth of 220 GHz at 3.4 THz with
∼10 μW per line.
The speed of the new system could
make it useful for real-time quality con-
trol of pharmaceutical tablets on a produc-
tion line and other fast-paced processes.
“Imagine that every 100 microseconds a
tablet is passing by, and you can check if
it has a consistent structure and there’s
enough of every ingredient you expect,”
professor Gerard Wysocki said.
As a proof of concept, the researchers
A new imaging technology rapidly measures the chemical compositions of solids. A conventional image
created a tablet with three zones contain- of a sample pill (left). Looking at the same surface with terahertz frequencies reveals various ingredients
ing common inert ingredients in pharma- shown as different colors (right). Such images could aid quality control and development in pharmaceuti-
ceuticals: forms of glucose, lactose, and cal manufacturing, as well as medical diagnosis and treatment. Courtesy of Sterczewski et al.

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919BI_BioScan.indd 11 9/5/2019 7:56:47 AM

 b BIOSCAN

cooling to a low temperature, hindering

its practical application. Many researchers
are now working on lasers that will po-
tentially operate at room temperature. The
Princeton team said its dual-comb hyper-
spectral imaging technique will work well
with these new room-temperature laser
sources, which could then open many
more uses for the THz imaging device.
Because it is nonionizing, THz radia-
tion is safe for patients and could poten-
tially be used as a diagnostic tool for skin
cancer. The technology’s ability to image
metal could be applied to testing airplane
wings for damage after they are struck by
an object in flight.
The research was published in Optica,
Gerard Wysocki (left), associate professor of electrical engineering, and Jonas Westberg, associate a publication of OSA, The Optical
research scholar, helped create a new terahertz imaging system that represents a major step toward Society (https://doi.org/10.1364/OPTI-
developing portable scanners that could rapidly measure molecules in pharmaceuticals, or classify tissue CA.6.000766).
in patients’ skin. Courtesy of David Kelly Crow.

Light-activated dynamic looping triggers gene expression

PHILADELPHIA — Using a technique
called light-activated dynamic looping,
or LADL, scientists at the University
of Pennsylvania are investigating the
role that genome folding plays in gene
expression. The team has demonstrated
how LADL can be used for quickly creat-
ing specific genome folding patterns on
demand, using light as a trigger.
LADL combines aspects of two other
biotechnological tools — CRISPR/Cas9
and optogenetics. By using CRISPR/Cas9
to target the ends of a specific genome
fold, or loop, and then using optogenetics
to snap the ends together like a magnet,
the researchers can temporarily create
loops between exact genomic segments in
a matter of hours.
Once CRISPR/Cas9 was found to
solve the targeting problem, the re-
searchers turned to optogenetics for
biological mechanisms that could bind
the ends of the loops together. They
selected proteins CIB1 and CRY2, found
in Arabidopsis, a flowering plant that is
a common model organism for geneti-
cists. CIB1 and CRY2 are known to bind

A modification of CRISPR/Cas9 allowed researchers to home

in on the desired sequences of DNA on either end of the loop
they wanted to form. If those sequences could be engineered
to seek one another out and snap together under the other
necessary conditions, the loop could be formed on demand.
Courtesy of the University of Pennsylvania.

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 together when exposed to blue light.
“Once we turn the light on, these
mechanisms begin working in a matter
of milliseconds and make loops within
four hours,” researcher Mayuri Rege
said. “And when we turn the light off, the
proteins disassociate, meaning that we
expect the loop to fall apart.”
Researcher Ji Hun Kim said that while
some DNA loops are formed slowly, many
form quickly, occurring within the span
of a second. “If we want to study those
faster-looping mechanisms, we need tools
that can act on a comparable timescale,”
he said.
The researchers tested the ability of
LADL to create the desired loops using
high-definition 3D genome mapping tech-
niques and were able to demonstrate that
the newly created loops were affecting
gene expression.
The relationship between looping and
genome function is poorly understood,
and the extent to which loops are dynamic
on short timescales remains an unan-
swered question, the researchers said.
To conduct experiments on how genome
structure configurations contribute to
genome function, techniques are needed
that can, on command, manipulate spe-
cific loops that are rapid, reversible, and
able to work on the target regions with a
minimum of disturbance to neighboring
sequences. The ability to engineer the
loops and mechanisms that determine the
timing and quantity of genome expression
means that researchers will be able to
mimic those mechanisms in experimental
conditions, making LADL a valuable tool
for studying the role of genome folding on
a variety of diseases and disorders.
“It is critical to understand the genome
structure-function relationship on short
timescales because the spatiotemporal
regulation of gene expression is essential
to faithful human development and
because the misexpression of genes often
goes wrong in human disease,” professor
Jennifer Phillips-Cremins said. “The en-
gineering of genome topology with light
opens up new possibilities to understand-
ing the cause and effect of this relation-
ship. Moreover, we anticipate that over
the long term, the use of light will allow
us to target specific human tissues and
even to control looping in specific neuron
subtypes in the brain.”
The research was published in Nature
Methods (https://doi.org/10.1038/s41592-
019-0436-5).
BioPhotonics • September 2019
919BI_BioScan.indd 13
Superresolution imaging method
aids in water decontamination
ITHACA, N.Y. — An imaging tech-
nique developed by Cornell University
researchers shows promise as a tool for
decontaminating water. The new approach
allows imaging of catalytic reactions at
the nanoscale in real time to help scien-
tists learn the optimal size and shape for
the most effective catalyst particles.
This competition-enabled, superreso-
lution imaging technique is applicable to
Xianwen Mao (left), first author of the paper,
a wide range of catalytic reactions. It is
and Peng Chen, the Peter J.W. Debye Professor of
based on the incorporation of competi- Chemistry, are pictured in the microscope room in
tion into a single-molecule fluorescence- Olin Research Laboratory. Courtesy of Rocky Ye/
detection scheme. The competition (a Cornell University.
nonfluorescent reaction) suppresses the
fluorescent reaction, allowing the fluores-
cent reaction to be measured and mapped. investigating a photoelectrocatalytic reac-
This process, in turn, provides informa- tion that is important for water decontam-
tion about the nonfluorescent reaction. ination on single photocatalyst particles.
The method is named “competition- They imaged the oxidation of hydroqui-
enabled imaging technique with super- none, a micropollutant found in water, on
resolution,” or COMPEITS. bismuth vanadate catalyst particles and
The researchers demonstrated it by discovered previously unknown behaviors
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9/5/2019 7:56:55 AM
 b BIOSCAN

of catalysts that helped render hydroqui-
none nontoxic.
“This highly generalizable technique
can be broadly applied to image various
classes of nonfluorescent systems, such
as unlabeled proteins, neurotransmitters,
and chemical warfare agents,” professor
Peng Chen said. “Therefore, we expect
COMPEITS to be a breakthrough technol-
ogy with profound impacts on many fields
including energy science, cell biology,
neuroscience, and nanotechnology.”
The research was published in Nature
Chemistry (https://doi.org/10.1038/
s41557-019-0288-8).

IR imaging technology could improve breast cancer detection

ROCHESTER, N.Y. — Researchers
at Rochester Institute of Technology
(RIT) and physicians from the Rochester
Regional Health System (RRHS) have
developed a noninvasive, cost-effective
method using IR technology to locate
hard-to-find breast cancer tumors. The
system consists of an IR camera on a
track mounted underneath a cushioned
table. The track is angled and can be ad-
justed as the clinician moves the camera
to take images. The team is also using
advanced computer simulation technol-
ogy to do predictive analysis on tumor
locations and growth.
The RIT-RRHS team received funding
from the National Science Foundation
(NSF) to screen patients and correlate IR
images against original MRI images, to
provide validation of the overall process (Left to right) Professor Satish Kandlikar and doctoral students Jose Luis Gonzalez Hernandez and
and technology. Alyssa Recinella discuss an artificial intelligence system that provides predictive analytics to determine
more information about the progression of disease. Courtesy of M. Cometa/RIT.
“Current screening modalities rely
heavily on digital mammography, but this
technique has shortcomings, particularly noninvasive, and cost-effective,” said mography. Further studies are needed to
in the significant subset of women who Dr. Pradyumna Phatak. “Our preliminary decide the best way to utilize this technol-
have dense breast tissue. Infrared imag- data suggest that it may be a very sensi- ogy in practice.”
ing using our technique is easy, quick, tive adjunct to routine screening mam-

Antibody-based probe developed for live-cell imaging in vivo

FORT COLLINS, Colo. — To expand
the toolbox of imaging in living cells,
researchers from Colorado State Univer-
sity and the Tokyo Institute of Technology
have developed a genetically encoded,
antibody-based probe that works with
specificity in vivo. Probes built from
antibodies were genetically fused with
mature fluorophores. When loaded into
cells, the probes bind and light up specific
targets, or epitopes, within the proteins
of interest as soon as the epitopes are
accessible. The probe, called the human
influenza hemagglutinin (HA) franken-
body, can light up in multiple colors HA-
tagged nuclear, cytoplasmic, membrane,
and mitochondrial proteins in diverse cell
types.
As if stitching new limbs on a body
— hence the name “frankenbody” — the
scientists took the binding regions of a
normal antibody and grafted them to a
different scaffold that remains stable in
A frankenbody labeling mitochondria. Courtesy
of Ning Zhao/Colorado State University.
live cells but retains the specificity of the
antibody. With the goal of making their
tool immediately useful, the scientists
designed their probe to work with the
classic HA tag. “For the longest time,
people have been looking at HA-tagged
proteins in fixed, dead cells. Now we can
image the dynamics of those proteins in
live cells,” professor Tim Stasevich said.
The scientists demonstrated several ap-
plications, including single-protein track-
ing, single-RNA translation imaging, and
amplified fluorescence imaging in zebra-
fish embryos. All of these experiments are
more challenging when using traditional
fluorescent protein tags, the team said.
The new probe could be a useful
complement to the green fluorescent
protein (GFP), a biochemistry tool and
subject of a Nobel Prize that involves

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919BI_BioScan.indd 14 9/5/2019 7:56:59 AM

 genetically fusing a light-up green tag to
a protein of interest. The GFP is limited
by its relatively large size and the time it
takes to fluoresce. With the new fran-
kenbody probe, the tag is smaller and
becomes fluorescent faster, so the activity
of a protein of interest can be captured in
real time.
“We’re interested in intracellular
antibodies because you can use them as
imaging reagents in a live cell,” Stasevich
said. “You don’t need a tag, like a green
fluorescent protein, because instead you
have this fluorescent antibody that will
bind to your protein that you want to
visualize.”
The versatility of the HA frankenbody
could make it a powerful tool for imaging
protein dynamics in vivo, the researchers
believe. Stasevich’s team is particularly
interested in studying RNA translation,
and they plan to use the new system to
more easily design new RNA imaging Frankenbodies (green) label nuclear proteins, mitochondria, stress fibers, and neuron membranes in
experiments. living cells. Courtesy of Ning Zhao/Colorado State University.
The research was published in Nature
Communications (https://doi.org/10.1038/
s41467-019-10846-1).

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 b BIOSCAN

Optochemogenetics tested for neuronal repair after stroke

ATLANTA — Researchers at the Emory
University School of Medicine have
developed optochemogenetics, a combina-
torial approach based on optogenetics and
chemogenetics, to enable the selective,
noninvasive stimulation of brain cells
using light. In a mouse model of stroke,
neural progenitor cells that received light
stimulation through optochemogenetics
promoted functional recovery in the mice.
The Emory investigators wanted to
remove the fiber optic cable that opto-
genetics approaches use to activate or
inhibit neurons. They created luminopsins
— engineered proteins that are both light
sensitive and able to generate their own
light when provided with coelenterazine
(CTZ). The researchers introduced genes
encoding luminopsins into stem cells,
which were cultured to form neural pro-
genitor cells. The neural progenitor cells
were delivered into the brains of mice
a week after stroke. CTZ was provided
intranasally twice a day for two weeks.
Bioluminescence could be detected in the
cell graft area and was visible for around
one hour after CTZ administration.
CTZ promoted an array of positive
Gaussia luciferase (GLuc) is fused to the channelrhodopsin (ChR) protein. ChR can be activated by blue
effects in the progenitor cells: more sur- light or by light emitted by GLuc when binding to its substrate coelenterazine (CTZ). YFP = yellow
vival and intact axons, more connections fluorescent protein. Courtesy of Shan Ping Yu.
within the brain, and better responses to
electrical stimulation. It also promoted
recovery of function in the affected limbs port of CTZ stimulation. The noninvasive
in the mice. In young mice, CTZ and repeated CTZ stimulation of transplanted
progenitor cells together could restore use cells is feasible for clinical applications,
of the stroke-affected limb back to normal the researchers believe.
levels, and in older mice they produced “It is not sufficient to put the cells into
partial recovery of function. the damaged brain and then not take care
The team believes that optochemo- of them,” Yu said. “If we expect pro-
genetics could represent a significant genitor cells to differentiate and become
advancement in light-based medicine. functional neurons, the cells have to
“Optogenetics is a fantastic technical tool, receive stimulation that mimics the kind
but it presents some barriers to clinical of activity they will see in the brain. They
implementation,” professor Shan Ping also need growth factors and a supportive As part of stroke research, Emory scientsts are
Yu, M.D., said. “You have the invasive environment.” making neural progenitor cells glow inside the
Yu and colleagues are also testing their brain. Bioluminescence from cells cultured in a
fiber optic light delivery, and the limited
multielectrode array next to a fiber optic cable.
distance of light diffusion, especially on approach for the delivery of neural pro- Courtesy of Jack Tung.
the larger scale of the human brain.” genitor cells in the context of traumatic
When designing experiments, delivery brain injury.
of cells into the brain for light activation The research was published in The
could offer scientists flexibility — in di- Journal of Neuroscience (https://doi.
rect light application, which can be turned org/10.1523/JNEUROSCI.2010-18.2019).
on and off quickly, or in the steady sup-

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919BI_BioScan.indd 16 9/5/2019 7:57:07 AM

 RAPIDSCAN Business and Markets

Prellis Biologics receives $8.7M, reports tissue printing progress

Prellis Biologics Inc., developer of a
holographic organ printing system, has re-
ceived an $8.7 million investment led by
Khosla Ventures. The investment is a Se-
ries A and comes after Prellis announced
major milestones in tissue engineering.
The company recently reported positive
results from the first animal transplanta-
tion of its 3D tissue scaffolds — called
Vascular Tissue Blanks — carried out
at Stanford University. The transplanted
structures were found to support human
tumor growth rates similar to or better
than typical tumor transplant methods, Prellis structure transplanted alone is surrounded by single-cell walled capillaries within two weeks
and with fewer cells. Typical tumor of transplantation into immunocompetent mouse (left). Human tumor cells transplanted in Prellis
studies in animals require 2 million or structures grow rapidly, are highly vascularized, and demonstrate minimal hypoxia (right)
(red = CD31, blue = DAPI, green = printed structure). Courtesy of Prellis Biologics.
more cells. With Prellis’ technology, full
tumor engraftment and vascularization
were achieved with just 200,000 cells, tial to significantly reduce overall animal Memorial Sloan Kettering Cancer Center
said Prellis co-founder and CEO Melanie use and speed up drug discovery efforts.” — are experimenting with the technology.
Matheu. Currently, more than 30 pharmaceuti- The company’s original seed round
“A breakthrough like this opens the cal and academic research labs — includ- investors include True Ventures and
door to studying rare human tumors and ing groups at the University of California, IndieBio, and they have joined Khosla in
complex human tumor immune system San Francisco; the University of Cali- the round. Total invested capital in Prellis
reactions,” Matheu said. “It has the poten- fornia, Irvine; Johns Hopkins; and the is currently at $10.5 million.

Olympus invites entries for Global Light Microscopy Image of the Year
Olympus has announced its first Global
Image of the Year Life Science Light
Microscopy Award, expanding upon its
established Image of the Year European
Life Science Light Microscopy Award.
Those interested in entering the contest
may do so through Jan. 31, 2020. Winners
of the contest will be selected by a jury
and announced in March 2020.
The jury consists of global representa-
tives from both science and the arts, in-
cluding photographer Ron Caplain; Geoff
Williams, a bioimaging facility manager
at Brown University; Urs Ziegler, the head
of a microscopy imaging facility at the
University of Zürich; Stefan Terjung, the
operational manager of an advanced light
microscopy facility at EMBL Heidelberg; The 2018 European Life Science Light Microscopy Award’s winning image was taken by Håkan Kvanström
Hiroaki Misono, a graduate school profes- and shows the shell of a marine snail covered in algae and cyanobacteria. Courtesy of Olympus.
sor of brain science at Doshisha Univer-
sity; Zhu Xueliang, a professor at the
Shanghai Institute of Biochemistry and Three images may be uploaded per awarded in addition to the global prize.
Cell Biology, Chinese Academy of Sci- contestant along with a brief explanation, Participants can win a CX43 microscope
ences; Yalin Wang, director of Biomedi- including the equipment used. Entries with a DP27 digital camera, X Line objec-
cal Research Core Facilities at Westlake will be evaluated based on artistic and tives, or an OM-D E-M5 Mark II camera.
University, Hangzhou, China; and Wendy visual aspects, scientific impact, and For more information or to enter, visit
Salmon, a light microscopy specialist of a microscope proficiency. Regional prizes www.olympus-lifescience.com/ioty.
bioimaging facility at MIT. in Asia, Europe, and the Americas will be

BioPhotonics • September 2019 17

919BI_RapidScan.indd 17 9/5/2019 8:30:14 AM

 r RAPIDSCAN

Precision Optics acquires portion of Ross Optical for $2M

Precision Optics Corp. Inc., a designer
and manufacturer of advanced optical
instruments for the medical and defense
industries, has completed the acquisition
of certain assets of Ross Optical Indus-
tries Inc. for up to $2 million.
The total purchase price comprises
$1.5 million in cash at closing and addi-
tional payments up to $500,000 subject to
a three-year earnout provision. Precision
Optics, headquartered in Gardner, Mass.,
completed an equity capital raise of
$950,000 at a per share price of $1.25 con-
current with the closing of the acquisition.
Ross Optical, based in El Paso, Texas,
had revenues of $3.9 million and net in-
come of $410,000 for the 12-month period
that ended Dec. 31, 2018. As part of the
transaction, Precision Optics is acquiring
slightly over $1 million of net working
capital.
Divi Mangadu, who has been Ross
Optical’s long-standing president, will
continue in that leadership role in the
company’s existing facility in El Paso.
Incorporated in 1989, Ross Optical
evolved from an optical components sup-
plier into an expanded solutions provider
with a portfolio of optical fabrication and
quality assurance equipment, technology,
and technical solutions that supply a num-
ber of industries, including the defense,
medical, and industrial markets.
Precision Optics has been developing
and manufacturing advanced optical
instruments since 1982. The company’s
medical instrumentation line includes
endoscopes and endocouplers, as well
as custom illumination and imaging
products for use in minimally invasive
surgical procedures.

SCANLAB appoints
Sonner R&D director

Christian Sonner, SCANLAB’s newly appointed Thomas Hümmer (center) was awarded €6000 ($6728) for the development of a highly sensitive
head of R&D. Courtesy of SCANLAB. microscope. Courtesy of the Center for NanoScience.

SCANLAB GmbH, an OEM manufac- Doctoral student wins Nano Innovation

turer of laser scan systems, has appointed
Christian Sonner head of research and
development to strengthen the company’s
market position in digital products and
control solutions for the photonics indus-
try.
Sonner, who has extensive expertise in
developing system solutions, is expected
to increasingly focus R&D on highly in-
tegrated scan solutions and drive forward
interdisciplinary collaboration.
Since 2013, Sonner was the company’s
software development manager.
SCANLAB has been developing and
manufacturing galvanometer scanners
and scan solutions since its founding in
1990. Its products turn lasers into precise
and flexible tools that provide the basis
for performing processing tasks.
Award for highly sensitive microscope
Thomas Hümmer, a doctoral student at the Ludwig-Maximilians-Universität (LMU)
Munich and Max Planck Institute of Quantum Optics, has been awarded €6000 ($6728)
for developing a highly sensitive microscope that explores the optical properties of nano-
objects. The Nano Innovation Award is given for best innovative doctoral thesis.
Hümmer’s microscope uses two opposing mirrors whose light is reflected back and
forth hundreds of thousands of times. If a nanoparticle is placed between the mirrors, its
interaction with light will be strongly enhanced. This effect allows the detection of light
absorption as weak as one photon in a million.
Because one of the mirrors is the size of a human hair, a scanning microscope can be
built that enables highly sensitive imaging and spectroscopy of tiny structures, which
could be useful for material research, nanotechnology, and the life sciences.
Hümmer has already developed a portable, fully functional prototype of the new
microscope. He is currently working on commercializing his findings and has started a
company named Qlibri.
The LMU Center for NanoScience (CeNS) awarded the Nano Innovation Award to-
gether with four spinoff companies from CeNS: attocube systems, ibidi, Nanion Tech-
nologies, and NanoTemper Technologies.
CeNS is a scientific institution of LMU Munich that promotes and coordinates inter-
disciplinary research in the field of nanoscience.

18 BioPhotonics • September 2019

919BI_RapidScan.indd 18 9/5/2019 8:30:17 AM

 Agilent Technologies TOPTICA Photonics
to acquire BioTek Instruments opens facilities
Agilent Technologies Inc. — a research, development, and manufacturing company —
has agreed to pay $1.2 billion to acquire BioTek Instruments Inc., a maker of cell imaging
in China
systems.
Headquartered in Winooski, Vt., BioTek designs, manufactures, and distributes life
science instrumentation including cell imaging systems, microplate readers, washers,
dispensers, automated incubators, and stackers.
Agilent entered the cell analysis market in 2015 with the acquisition of Seahorse
Bioscience, a provider of specialized instruments and live-cell, kinetic assays. Agilent
Seahorse XF technology was created to evolve cellular metabolism analysis, allowing
researchers to better understand metabolic profiles in live cells.
In January 2018, Agilent acquired Luxcel Biosciences. Luxcel’s assays use soluble
sensors to analyze metabolism, complementing Agilent’s Seahorse XF technology and
providing researchers with more options to analyze live-cell metabolism.
In September 2018, Agilent acquired ACEA Biosciences Inc., a developer of high-
performance cell analysis platforms for life science research.
Upon closing of the BioTek acquisition, the size of Agilent’s cell analysis business will
be in excess of $250 million in annual revenues.

6.2%
— expected compound annual
growth rate of the photodynamic
therapy market by 2029, according
to Persistence Market Research
Five years ago, BioPhotonics reported the func-
tional use of diffuse optical tomography (DOT),
a technique that had previously been very limited
in its application. The technique allowed for the
imaging of brain activity without radiation. Since
then, researchers have developed DOT further,
bringing its resolution and effectiveness closer
to that of magnetic resonance imaging, which
remains the gold standard of brain imaging.
Courtesy of TOPTICA.
Laser developer and producer TOPTICA
Photonics has expanded into China with
a head office in Shanghai and a branch
office in Beijing to provide sales, service,
and application support for its full range
of technologies.
TOPTICA Photonics China was
established to serve the needs of the
Chinese academic and industrial sectors
in the markets of quantum technologies,
biophotonics, materials processing, and
test and measurement, and joins offices in
Germany, the U.S., and Japan.
Juergen Stuhler, chairman of the board
of directors and general manager of TOP-

2014
The method has been in the
works for more than a decade,
but until recently, it was limited to small regions
of the brain. Now DOT can cover two-thirds of the
head, imaging brain processes in multiple regions
and networks, including those involved
with language processing and daydreaming.
— BioPhotonics Bioscan, September 2014
TICA Photonics China, said the company
can now offer customers in China direct
sales and services for picosecond and
femtosecond seeders.
“We now have the ability to support
and supply customers in the areas of
quantum technology, tunable and single-
frequency diode lasers, frequency combs,
wavemeters, light engines for laser light
microscopy, and terahertz systems for
industrial metrology and security applica-
tions,” he said.
TOPTICA was founded in 1998 near
Munich and built its reputation on deliv-
ering high-end specifications.

BioPhotonics • September 2019 19

919BI_RapidScan.indd 19 9/5/2019 8:30:21 AM

 r RAPIDSCAN

Juergen Czarske receives OSA award

The Optical Society (OSA) has named Juergen Czarske of Technische Universität Dres-
den (TU Dresden) the recipient of the 2019 Joseph Fraunhofer Award/Robert M. Burley
Prize for seminal contributions to the field of digital interferometric and holographic
sensors.
OSA President Ursula Gibson said, “Czarske’s achievements in optical engineering
have led to significant innovation across several fields, including important biomedical
applications.”
Czarske’s research involves laser-based metrology for use in biomedicine, particularly
for neurodegenerative diseases.
Czarske is director and C4 professor at TU Dresden; life fellow of OSA and SPIE; fel-
low of EOS; senior member of IEEE; elected member of the Saxon Academy of Sciences
and the Scientific Society for Laser Technology; and board member of the German Soci-
ety of Applied Optics (DGaO), the German branch of the EOS, and the German Associa-
tion for Laser Anemometry (GALA).
Czarske’s interests include unconventional optical imaging with deformable mirrors,
wavefront shaping for viewing through scattering media, and optogenetics with human- Courtesy of Juergen Czarske.
induced pluripotent stem cell-derived neural networks.
Czarske has received the Berthold Leibinger Innovation Prize, the Reinhart Koselleck
Project of the German Research Foundation (DFG), the Measurement Technique Award
of AHMT, and many other honors. He has published about 200 journal articles, has over
20 patents, and has delivered more than 100 invited lectures.

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 EN E S T EDT. S E
BioTek receives
business growth award
BioTek Instruments Inc. was among the businesses that received
a Vermont Business Growth Award on Sept. 17. The awards are
presented annually by Vermont Business Magazine and Key-
Bank, acknowledging the top 25 fastest-growing businesses in
the state.
BioTek has shown 67.4% growth over the past five years. It
was recognized within the technology category at the award
ceremony. This was the seventh time the company received the
award.

$48.6B
— size of the global medical
imaging market by 2025, according
to Zion Market Research

Spectral Evolution
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919BI_RapidScan.indd 21 9/5/2019 8:30:26 AM

 A multicolored retinal blood oxygen saturation map overlaid on a VIS-OCT angiogram. Adapted from Reference 5.

VIS-OCT Opens Eyes to New Approaches

Attention and investment could revolutionize applications
in visioning and treatment of eye-related diseases.

BY HAO F. ZHANG, NORTHWESTERN UNIVERSITY, VIS-OCT Publications VIS-OCT Research

AND KIEREN PATEL, OPTICENT HEALTH

F
irst reported in 1991 by Huang and
colleagues1, OCT has become an
important imaging tool for ophthal-
mology following innovations over the
past three decades. OCT noninvasively
provides 3D in vivo optical biopsy with
microscopic resolution in the eye at a
depth range up to the choroid2. Besides
3D anatomical imaging, OCT can also
provide functional information. The most
Figure 1. Number of scientific publications on VIS-OCT. Data obtained from Web of Science
notable functional extension is OCT
and Google Scholar (a). Annual NIH research support for VIS-OCT awarded by the end of 2018.
angiography (OCTA), which can map Data obtained from NIH RePORT (b).
the microvasculature in the eye without
contrast agent and is being investigated in
clinical ophthalmology. izing the complete vascular network, importantly, OCTA is compatible with
OCTA opens up a new window on bet- including the multilayer inner retinal most existing OCT systems and can be
ter managing a wide variety of blinding capillary network and deep choroidal ves- implemented through a software upgrade.
diseases through noninvasively visual- sels, without using extrinsic labels. More All commercial OCT systems, to

22 BioPhotonics • September 2019

919BI_Feat VISOCT.indd 22 9/5/2019 8:16:53 AM

 date, use NIR light as a result of vari-
ous economical, clinical, and technical
considerations. However, as broadband
visible light sources generating low-
coherence light from 400 to 2000 nm or
more have become available, visible-light
OCT (VIS-OCT) device development
has gained momentum, as evidenced by
the dramatically increasing number of
scientific publications and presentations
(Figure 1a). There are now many research
groups from institutions around the
world — such as the Medical University
of Vienna; the University of Amsterdam;
Shanghai Jiao Tong University; the Uni-
versity of California, Davis; and Oregon
Health & Science University (OHSU) —
devoted to various aspects of VIS-OCT
developments, from optical engineering
to signal processing and clinical tests.
One startup company, Opticent Health,
has also started to commercialize VIS-
OCT for both preclinical and clinical
usages.
Figure 1a shows the total number of
scientific publications from 2013, when
VIS-OCT was first reported by the Zhang
Lab for retinal oximetry in rodents3, to
the end of 2018. Figure 1b shows the
annual research funds awarded by the
National Institutes of Health (NIH) to
conduct various investigations using VIS- Figure 2. VIS-OCT retinal imaging in mouse. En face fundus image (a). Microangiogram by VIS-OCT
OCT. In 2019 alone, over $5 million has (b). A high-definition B-scan image showing all the fine details of the retinal layers (c). Reprinted and
been awarded for VIS-OCT research, and adapted from Reference 5.
NIH investment has steadily increased
since 2014. It is clear that VIS-OCT has
the potential to become mainstream. One by Opticent Health. Using a bandwidth Figure 2a shows a typical VIS-OCT
of the original OCT inventors, OHSU of 100 nm, ultrahigh axial resolution amplitude fundus image of a wild-type
professor David Huang, recently com- can be achieved, which well exceeds the mouse retina. The radial pattern of major
mented that VIS-OCT is “a new frontier” state-of-the-art NIR OCTs. The reported blood vessels and the capillary bed are
for OCT research4. illumination power of VIS-OCT on hu- clearly visualized in the fundus image as
man studies is 220 µW or less, which is a result of the strong optical absorption of
Imaging in mouse, human eyes considered eye-safe according to ANSI visible light by hemoglobin. When taking
The motivations for VIS-OCT develop- Z136.1 (2014). advantage of the motion contrast of the
ment came primarily from the demand
for higher spatial resolution and new
imaging contrasts to improve disease Table 1. Key Parameters of the Latest VIS-OCT System
management at ever finer tissue levels
and beyond anatomical alterations. With Parameters Values
comparable bandwidth, visible-light
illumination significantly improves the A-Line Rate Up to 250 kHz
OCT axial resolution over and above NIR
Spectral Range 510 to 610 nm
light. In addition, VIS-OCT can retrieve
unique scattering and absorption contrasts Axial Resolution in Tissue ~1 µm
from the ocular tissue within the visible
spectral range to extract new functional Illumination Power on the Cornea 220 µW
information, such as retinal hemoglobin
oxygen saturation3. System Roll-Off 5 dB/mm
Table 1 summarizes the key param-
eters of the VIS-OCT system developed Courtesy of Opticent Health.

BioPhotonics • September 2019 23

919BI_Feat VISOCT.indd 23 9/5/2019 8:16:56 AM

 Visible-Light OCT

Figure 3. VIS-OCT B-scan image of human retina. ELM: external limiting membrane. Courtesy of Ian Rubinoff/Northwestern University.

blood, VIS-OCT angiogram (VIS-OCTA)
can be achieved in the same manner as
the modern OCTA. The angiographic
image from the same volume as Figure
2a is shown in Figure 2b. All three layers
of the capillaries are clearly visualized
at the single capillary level. Figure 2c
shows a high-definition B-scan image
from a mouse eye, where all of the micro-
anatomic layers are clearly imaged with
ultrahigh axial resolution.
Researchers from UC Davis and
Northwestern University have ap-
plied VIS-OCT to image human eyes
to measure retinal hemoglobin oxygen
saturation and ultrafine inner and outer
retinal anatomical sublayers (sidebar).
Figure 3 shows a high-definition B-scan
image from a healthy human eye. Figure
3a shows the image from the fovea, and
Figure 3b shows the magnified view of
the area highlighted by a red square. All
five outer retinal layers — including the
inner/outer segment junction (IS/OS), the
cone outer segment tips (COST), the rod
outer segment tips (ROST), the retinal
pigment epithelium (RPE), and Bruch’s

Figure 4. Method to extract sO2 from single retinal vessels (a). Pseudo-colored sO2 map overlaid on VIS-OCT angiogram (b). En face image of segmented RNFL/
GCL (c). Spectroscopic VIS-OCT image stack (d). Images (a) and (c) adapted from Reference 3; (b) adapted from Reference 5; (d) adapted from Reference 9.

24 BioPhotonics • September 2019

919BI_Feat VISOCT.indd 24 9/5/2019 8:17:01 AM

 membrane (BM) — are clearly resolved.
These minute outer retinal anatomical Seeing Potential in Retinal Imaging
layers are so far not easily resolvable by
NIR-OCTs without being enhanced by In 2015, the Zhang group from Northwestern University and Dr. Amani Fawzi,
adaptive optics. a Northwestern ophthalmologist, reported the first demonstration of human reti-
nal imaging using VIS-OCT. The authors showed that VIS-OCT provides much-
Clinical and preclinical uses improved imaging contrast and axial resolution in visualizing fine anatomical
Besides ultrahigh axial resolution, features — of retinal nerve fiber layers and lamina cribrosa, for example — as
another key advantage of VIS-OCT is compared with modern clinical OCTs.
its sensitivity to new tissue scattering In 2017, the same Northwestern team reported the measurement of retinal
and absorption properties in the visible oxygen saturation in humans. In that work, the authors imaged healthy volun-
spectral range. These added sensitivities teers and showed that oxygenation levels decrease with arterial vessel bifur-
enable VIS-OCT to explore new applica- cations after applying a statistical denoising algorithm. In the same year, the
tions for clinical management of several Srinivasan group from the University of California, Davis successfully measured
blinding diseases. retinal oxygen saturation in humans, together with blood flow and hematocrit.
In the fi rst example, several groups Both the Northwestern and UC Davis teams developed spectroscopic analyses
have applied VIS-OCT to quantitatively to extract blood oxygenation from the full-band VIS-OCT image. Prior to these
measure retinal blood oxygen saturation two reports, only fundus photography-based methods were reported for retinal
(sO2). It is believed that better under- oximetry, where lack of axial resolution leads to challenging repeatability. Reti-
standing of retinal oxygen metabolism nal oximetry is believed to be greatly beneficial in managing diabetic retinopa-
could greatly facilitate the diagnosis6 thy, macular degeneration, and glaucoma.
and anti-VEGF (antivascular endothelial Recently, the Northwestern and UC Davis teams started to use the quality of
growth factor therapy) treatments of VIS-OCT retinal imaging and VIS-OCT’s ultrahigh axial resolution for clinical
diabetic retinopathy and macular degen- management of glaucoma, for example. The UC Davis team corrected the chro-
eration7,8. Figure 4a shows the process matic aberration of VIS-OCT illumination and further manipulated the spectral
of spectroscopic analysis of VIS-OCT profiles of VIS-OCT probing light to achieve nearly submicron axial resolution.
signals to calculate sO2. For each 1D The Northwestern team developed a speckle reduction method through spatial
interferogram, researchers divide the full beam modulation and demonstrated significantly improved imaging quality
spectrum into several subbands (Figure in resolving minute anatomical features in both the inner and outer retinas. In
4a, top). Then VIS-OCT reconstructs addition, the Yi group from Boston University further integrated NIR light OCT
B-scan images in each subband to form with VIS-OCT through an achromatizing lens for dual-band retinal imaging,
the basis of spectroscopic analysis. The aiming to detect spectral signatures of the retinal nerve fiber layer under vari-
bottom of Figure 4a shows the B-scan ous stages of glaucoma.
images of a retinal vessel from differ-
ent subbands using short-time Fourier
transform. These spectral signatures can
be correlated with optical absorption
of both oxygenated and deoxygenated
hemoglobin to estimate sO2. As shown in
Figure 4b, sO2 in each individual major
retinal blood vessel can be extracted. We
measured the mean sO2 values in arteries
and veins at 95 ± 3% and 72 ± 7% in a
mouse, respectively5.
A second example is VIS-OCT’s
capability to potentially diagnose differ-
ent types of glaucoma far earlier — for
example, through spectroscopic sensing
of neuron damages — than clinically
detectable biomarkers currently can.
Figure 4c shows the en face image of
the retinal nerve fiber layer (RNFL) and
ganglion cell layer (GCL), where retinal
ganglion cell axon fiber structures are
clearly visualized as a result of the
much-improved contrast in the visible-
light spectral range. Using a similar
short-time Fourier transform-based
spectroscopic analysis of the RNFL Figure 5. Treatment image. Courtesy of Opticent Health and Joel Schuman/Langone Medical Center,
(Figure 4d), VIS-OCT can provide NYU School of Medicine.

BioPhotonics • September 2019 25

919BI_Feat VISOCT.indd 25 9/5/2019 8:17:06 AM

 Visible-Light OCT
insight into ultrastructural damage well
before RNFL thinning occurs9.
VIS-OCT represents one of the most
exciting new imaging directions for
clinical ophthalmology. With superior
resolution and the ability to generate
accurate functional information, VIS-
OCT is likely to change diagnostic and
prognostic treatment protocols for a
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919BI_Feat VISOCT.indd 26
variety of blinding diseases. More OCT light source; thus, new light sources are
research groups and physician scientists needed through continuous technology
are needed to explore its full potential. In innovations.
addition, the cost of VIS-OCT systems
needs to come down to be comparable Meet the authors
to existing commercial OCTs and to Hao F. Zhang, Ph.D., is professor of biomedical
facilitate broad clinical adoption. Reduc- engineering and ophthalmology at North-
ing the price of VIS-OCT is predomi- western University. His lab focuses on new
nately constrained by the high cost of its technologies of optical coherence tomography
and single-molecule imaging, as well the
application of these technologies to biology
and medicine. He received his doctoral degree
in biomedical engineering from Texas A&M
University and postdoctoral training at Wash-
ington University in St. Louis.
Kieren Patel (Ph.D., MBA, J.D.) is CEO of
Opticent Health, which seeks to commercial-
ize visible-light optical coherence tomography
for both clinical and preclinical markets. He
co-founded Opticent Health with professor
Hao F. Zhang in 2015 in Evanston, Ill. Patel re-
ceived his doctorate in molecular biology from
the University of California, Berkeley and his
MBA and J.D. from Northwestern University.
X-Cite® XYLIS
Broad Spectrum LED Illumination References
1. D. Huang et al. (1991). Optical coherence
• A true arc lamp replacement for
tomography. Science, Vol. 254, Issue 1178.
the widest range of imaging
2. W. Drexler and J.G. Fujimoto (2015). Optical
applications
Coherence Tomography: Technology and
• Our highest power LEDs for Applications. N.Y.: Springer.
fluorescence on compound and 3. J. Yi et al. (2013). Visible-light optical
stereomicroscopes coherence tomography for retinal oximetry.
Opt Lett, Vol. 38, pp. 1796-1798.
4. S. Binder (Jan. 2019). Ophthalmic imag-
ing modalities: a lot of change in 2 years.
X-Cite mini+™ EyeWorld, www.eyeworld.org/ophthalmic-
Compact
Compacct LE
Co Illumination
LED Illu
umination
on imaging-modalities-lot-change-2-years.
• Perfect mercury-free option for 5. S. Chen et al. (2015). Measuring oxygen sat-
routine imaging applications uration in retinal and choroidal circulations
in rats using visible light optical coherence
• Compact and economically tomography angiography. Biomed Opt Exp,
priced Vol. 6, pp. 2840-2853.
6. R.A. Linsenmeier and H.F. Zhang (2017).
Retinal oxygen: from animals to humans.
USB, TTL, Optional Light
DAPI Direct Prog Retinal Eye Res, Vol. 58, pp. 115-151.
Cy7 speedDIAL foot guide
to Cy5 coupled 7. P.L. Nesper et al. (2017). OCT angiography
control pedal coupled
and visible-light OCT in diabetic retinopa-
• • • •
thy. Vision Res, Vol. 139, pp. 191-203.
• • •
8. W. Liu et al. (2017). Increased inner retinal
oxygen metabolism precedes microvascular
alterations in rodent model with Type 1
diabetes. Invest Ophthalmol Vis Sci, Vol. 58,
Society for Neuroscience
October 20 - 23, 2019 pp. 981-989.
McCormick Place | Chicago, IL 9. J. Yi et al. (2016). Optical detection of early
Booth #910 damages in retinal ganglion cells in a mouse
model of partial optic nerve crush injury.
Invest Ophthalmol Vis Sci, Vol. 57, pp. 5665-
5671.
BioPhotonics • September 2019
8/23/19 9:32 AM
9/5/2019 8:17:09 AM
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 Lens-Free Microscopy
Focuses on Commercialization
Years after Aydogan Ozcan’s demonstration, the technology
evolves to meet real-world applications.

BY HANK HOGAN, CONTRIBUTING EDITOR

W
ith advancements in image
processing and sensors, lens-
free microscopy is ready for
a closer look by researchers, industry,
and consumers. Compared to tradi-
tional benchtop microscopes, lens-free
techniques can cost less, reduce instru-
ment size, enlarge the field of view, and
improve throughput.
“The device [a lens-free microscope] is
10× smaller than a regular microscope.
But the image, on the contrary, is 100×
larger than any [other] microscope image,”
said Cédric Allier, a project manager
with the Grenoble, France-based research
organization CEA-Leti.
The large field and depth of view makes
it possible to look at perhaps 10,000 cells
at once. Therefore, the lens-free approach
is well suited to needle-in-a-haystack
problems, such as detecting cancer or
other diseases. The small size, low cost,
and ruggedness of lens-free microscopes
also make them useful for hand-held
and portable devices that are capable of
detecting medical problems or performing
environmental monitoring in the field.

Above: Aydogan Ozcan displays hand-held, lens-free microscopes. The black one (left) is installed on a phone and uses the phone’s CMOS imager for lens-free
microscopy of a specimen. The white prototype (right) is a stand-alone hand-held lens-free microscope that is controlled by a laptop using a USB cable. This
stand-alone version weighs less than 50 g. Courtesy of Ozcan Lab/UCLA.

28 BioPhotonics • September 2019

919BI_FEAT Lens Free.indd 28 9/5/2019 8:15:45 AM

 A lens-free microscope consists of a semicoherent light source illuminating an object that sits directly atop an image sensor, a design that gives rise to holograms
that can be reconstructed into images. Courtesy of CEA-Leti.
Several small companies and startups
are either actively producing lens-free
products or plan to do so soon. If lens-free
methods offer the cost, performance, and
usability necessary to achieve commercial
success, these products promise to boost
public health by identifying waterborne
toxins, airborne pollutants, and infectious
diseases in the field.
How technology has changed
In a conventional microscope, light
interacting with a target is collected and
focused by lenses. Lens-free imaging,
as the name implies, eliminates such
bulk optics. Instead, based on a concept
that first appeared in papers co-authored
by Aydogan Ozcan, a UCLA electrical
engineering and bioengineering professor,
these on-chip microscopes have a light
source located centimeters from a speci-
men that sits directly atop a CMOS image
sensor.
The light is partially coherent, giving
rise to an interference pattern captured by
the sensor. Processing this holographic
data using an appropriate algorithm de-
signed specifically for the task creates an
image suitable for viewing and analysis.
The software also makes it possible to
focus digitally rather than mechanically.
A key to making this setup work is the
image sensor, according to Allier, who
has been working on lens-free techniques
for a decade. He said the pixel size of the
sensor helps determine the resolution of
BioPhotonics • September 2019
919BI_FEAT Lens Free.indd 29
the microscope. In 2010, readily available
image sensors had a roughly 5-µm pixel
size. By 2015, this had dropped to 1.7 µm,
making it possible to image cells. The
pixel size continues to shrink and is now
as small as 0.8 µm.
What’s more, over the last few years,
increasingly powerful GPUs, or graph-
ics processing units, have shortened the
image processing time. A half decade ago
it may have taken an hour to do this pro-
cessing for a 2D image, Allier recalled.
“Today it takes less than 30 seconds,
thanks to the GPU and the fancy algo-
rithm we’ve developed,” he said. This
speed makes 2D lens-free microscopy
acceptable for clinical work.
These microscopes use a monochro-
matic, narrowband LED that shines
through a 100-µm diameter pinhole, in
CEA-Leti’s version. This source is coher-
ent enough without the cost or power
requirements of a laser.
IPRASENSE, a company based in
Montpellier, France, was founded to
commercialize the CEA-Leti technology,
including the holographic reconstruction
algorithms, acquisition setup, and soft-
ware as well as the data analysis chain.
The almost 7-year-old company has a line
of cell-culture monitoring equipment that
uses lens-free microscopy and automated
algorithms to rapidly count cells and
determine their viability.
The automated approach is about
10× faster than the manual equivalent,
Allier said. Tests have shown the two
methods provide comparable results, and
the automated approach doesn’t require
skilled personnel in its operation.
After their initial papers on lens-free
imaging, Ozcan’s UCLA group produced
a 2010 Optics Express paper describing
much better resolution results than those
set by pixel size. The investigators did
this by shifting the aperture, and there-
fore the illumination center, in small steps
across the object. They then combined the
shifted holograms into the final image.
“This method computationally divides
each pixel into smaller pixels to be able
to reach the diffraction limit of light,”
Ozcan said. He added that later work
showed shifting the sensor achieved the
same effect.
This pixel-segmenting technique does
require the capturing and processing of
more data, and a more complex micro-
scope than otherwise would be the case.
But in addition to offering higher resolu-
tion, the stepped approach also reduces
speckle noise that arises from wavelength-
scale object surface roughness and
reduces unwanted reflection interference,
improving image quality.
Marketplace applications
Ozcan’s group has explored a range of
possible microscopy applications, includ-
ing portable units that count and classify
algae in seawater. These could provide a
faster and more field-friendly means to
29
9/5/2019 8:15:49 AM
 Lens-Free Microscopy

have ways to reconstruct lens-free

holograms more efficiently and rapidly
than before. Machine learning techniques
recently led to lens-free holographic
imaging with the color and contrast of a
light-field microscope, something not pos-
sible previously, Ozcan said.
He noted that lens-free microscopes
can be made for under $50, excluding the
computer used for processing. The device
weight can be less than 50 g, with a size
small enough to fit in the hand. Impor-
tantly, there are no lenses to align and no
focus to adjust, making the microscope
more useful in field applications.

A focus on the future

In addition to the long-time lens-free
A lens-free microscope that performs pixel-superresolution holographic on-chip lens-free microscopy in a microscopy researchers, there are some
hand-held design. It can image phase and amplitude information of transmissive samples (such as tissue relative newcomers to the field. For ex-
samples, cells, pathogens, etc.) with a resolution that is better than the size of each pixel of the CMOS ample, Euan McLeod is now an assistant
imaging chip. Courtesy Ozcan Lab/UCLA. professor of optical sciences at the Uni-
versity of Arizona in Tucson who worked
in Ozcan’s group at UCLA. McLeod
detect algal blooms, which would be a ing environmental monitoring and water is investigating lens-free microscopy
public health benefit, as such overgrowths analysis,” Ozcan said. applications that push the boundaries of
can create toxic conditions that shutter Sales of Lucendi products should start sensitivity and seek to detect very small
beaches. in 2019, he added. In 2018, CEA-Leti objects, such as viruses or individual
The technology could be used to deter- licensed a core patent from Cellmic, and protein molecules.
mine the presence of airborne pollen, mold this technology is now working its way This might be done, he said, via func-
spores, and other allergens, information into more products and applications. tionalized beads. Coating these micro-
important to allergy sufferers. It could also The arrival of open-source machine scopic particles with biomarkers that
be used to screen for parasites and other learning platforms such as TensorFlow is visibly react to a specific microorganism,
diseases, in the field and at the point of use. another recent innovation that is advanc- virus protein, or DNA strand makes it
To commercialize the technology, ing these technologies and their applica- possible to image tiny targets. This ap-
Ozcan spun off two companies, Cellmic tions, Ozcan said. With machine learning, proach could be used to observe and count
LLC and Lucendi Inc. Both are based in software is presented with the raw data biomarkers for detection of diseases,
Los Angeles. and the desired outcome in example cases including cancer.
“Cellmic has been focusing on point- where the final image is known. It then Screening tissue slices for cancer
of-care diagnostics and mobile health ap- figures out how to transform the data into requires viewing a very large number
plications. Lucendi has been focusing on the image. of cells at subcellular resolution. “You
high-throughput liquid analysis, includ- As a result, physicists and engineers need a very large field of view and at the

In lens-free imaging, computational algorithms combine low-resolution holographic data (left) to create a high-resolution hologram (center), which then
undergoes reconstruction into a microscopic image (right). Courtesy of Euan McLeod/University of Arizona.

30 BioPhotonics • September 2019

919BI_FEAT Lens Free.indd 30 9/5/2019 8:15:53 AM

 A prototype of a 3D lens-free video microscope for the monitoring of 3D cell culture. The specimen sits atop the image sensor chip (left).
A 3D reconstructed volume (4.7 mm3) of a 3D cell culture of prostatic cells (right). Courtesy of CEA-Leti.
same time good resolution, with ‘good’
meaning micron or submicron resolution,”
McLeod said.
Another recent entrant into lens-free
microscopy is imec, the Leuven,
Belgium-based research organization.
At the 2017 SPIE BiOS conference in
San Francisco, imec investigators
reported on the development of a lens-
free holographic microscope built using
a 1.12-µm-pixel sensor that achieved
micron-resolution imaging. However,
citing ongoing confidential research and
active work in this area, an imec spokes-
person said no further details or updates
were available.
The University of Arizona’s McLeod
noted that lens-free methods rival the
resolution possible with traditional
benchtop microscopes. In the past, light-
induced artifacts were an issue. However,
he said these resolution-robbing effects
can be eliminated by shifting the light
source location or its wavelength and
capturing multiple images. Software
can then combine the data and remove
artifacts, thereby improving image clarity
and usefulness.
BioPhotonics • September 2019
919BI_FEAT Lens Free.indd 31
McLeod noted that some imaging
methods present challenges to lens-free
microscopy. Fluorescence-based tech-
niques, for instance, can be problematic
because light emitted by the specimen is
incoherent with the illumination, thereby
destroying the interference that forms the
basis for holographic imaging.
Another issue that needs to be further
addressed is imaging speed. According
to CEA-Leti’s Allier, lens-free 3D imag-
ing today is too slow for anything but
demonstrations. It takes an hour, for ex-
ample, to complete the image processing
of a mosquito wing or other objects of
about 5 cubic millimeters volume. In the
future, faster GPUs should speed up this
process. Machine learning could also
lead to the development of new, more
efficient, and faster image reconstruction
algorithms.
There are several ways in which the
continuing evolution of lens-free micros-
copy could have an impact, particularly
in applications where the new technology
offers an advantage over traditional forms
of microscopy. On the clinical side, lens-
free methods may make it easier to detect
health problems, particularly infectious
diseases in the field. The new micro-
scopes, with their ability to rapidly and
efficiently screen cells, could also benefit
research and drug development.
On the consumer side, lens-free
microscopy could lead to hand-held
devices that enable people to measure and
monitor air quality by imaging airborne
particles, dust, mold, and allergens. The
microscopes could similarly monitor
water-quality issues. There have already
been demonstrations of such possibilities.
For example, Ozcan’s group reported, in
a 2010 Lab on a Chip paper, on a cell-
phone attachment that operates using this
technology.
According to Allier, further develop-
ments that improve device performance,
such as speeding up lens-free 3D imag-
ing, could spur new uses for lens-free
microscopy.
“This will open up new applications
that we haven’t thought of,” he said.
hank@hankhogan.com
31
9/5/2019 8:15:56 AM
 Fluorescence Microscopy:
Getting the Picture Right
Camera choice has to strike a balance between certain sensor properties, camera-related
topics, and the needs of the intended applications in industry or medicine.
BY FELIX ASCHE, BASLER AG
F
luorescence microscopy is a very
broadly used term covering numerous
applications. It ranges from basic life
science applications such as time-lapse
cell viability assays — in which dead
cells are counted as they start to fluoresce
by intrusion of a fluorochrome that cannot
enter intact living cells — to sophisticated
techniques where only very few photons
or single molecules are detected and
localized by specific high-end hardware
and software. It can be quite complex to
design an optical imaging system that
perfectly fits a specific application. And at
the same time, instrument manufacturers
are facing pressure to keep the costs down
in the medical and life sciences markets.
Optical format and resolution
Looking at the visible wavelength
range, the optical setup considerations
regarding format, magnification, and
resolution do not significantly differ from
normal light microscopy applications
and are therefore not described here.
However, it is important to know that the
Figure 1. Frontside-illuminated (left) and backside-illuminated pixels (right).
32
919BI_FEAT Flourescence.indd 32
overall costs increase when larger optical
formats such as the F-mount are used.
The most common mount is the C-mount,
which (with up to 1.1-in. sensors) delivers
very good optical performance, as well as
most products and solutions at a reason-
able price. The smaller S-mount is a good
choice when size-restricted and low-cost
instruments are developed. Squared sen-
sors are preferred to capture the maxi-
mum image content.
Because the sensor takes over a central
role in an imaging system, the selection
regarding certain performance specifica-
tions is of major importance.
CCD, CMOS, sCMOS, and BSI
From its beginning in the late 1970s,
CCD (charge-coupled device) technol-
ogy has been strongly established and is
still available in high-end microscopy
cameras. Sony, the leading manufacturer
of CCDs globally, has discontinued this
technology, and instrument manufactur-
ers using CCDs are being forced to find
alternative cameras for their systems.
Images courtesy of Basler AG.
The newer CMOS technology has spread
through consumer sensor markets for
many years and has recently become com-
petitive. Noise levels are now comparable
or even better than with the traditional
and commonly used CCD sensors, en-
abling the new technology to deliver
higher speed, higher resolution, and less
power consumption/heat dissipation at
lower prices into medical and life sciences
applications.
Scientific CMOS (sCMOS) cameras
were introduced in 2010 through the col-
laboration of three companies working
to provide better performance (mainly
through faster CMOS-like readout speeds
and higher dynamic range) compared to
aging CCD technology, which contin-
ued to provide a superior noise behavior
and brought higher image quality and
sensitivity. CMOS sensor development
is still rapidly accelerating. Recently, a
technology called backside illumination
(BSI) found its way into industrial im-
age sensors. BSI significantly improves
the pixels’ quantum efficiency. Coming
BioPhotonics • September 2019
9/5/2019 8:14:15 AM
 Figure 2. A persistent fluorescence carrier with 8.5-µm small structures of varying intensity was imaged under the same microscopic conditions (same optical path
and lighting, and 10-s exposure) with a current BSI (Basler ace acA3088-57um, IMX178) and FSI sensor (Basler MED ace 5.1 MP, IMX250). For the FSI camera,
the histogram shows nearly doubled gray values.

from the smartphone market with its of traditional CCD technology will tail is needed to calculate color information of
demand for smaller pixels, this technol- off. For instrument manufacturers and the image using a process called debayer-
ogy involved reversing the orientation of their current and future products, cameras ing. As the color filters block a certain
the pixel structure to present the light- with CMOS sensors are acknowledged as amount of light, fewer photons reach the
sensitive photodiode directly below the the better choice. photon-reactive area of the pixel. In addi-
microlenses. With this design the metal tion to the Bayer pattern on the sensors,
wiring structures no longer inhibit the Monochrome or color the IR-cut filter in color cameras presents
incident photons, improving the so-called For fluorescence applications, mono- a limiting factor because it blocks light
fill factor (the relation of the photoactive chrome cameras are usually preferred of approximately 650 to 700 nm upward
and nonphotoactive area) of the pixels because of the higher quantum efficiency. (Figure 3).
(Figure 1). The technical factor driving this dif- Typically, images with multiple fluores-
However, BSI sensors can have other ference is that in color cameras, Bayer cence markers for specific detection and
additional noise sources contributing microfilters on each pixel let only certain co-localization of molecules of interest
to the dark current. Dark current is the wavelengths pass through. This filtering are made from separate images using
leakage of electrons during exposure.
This can make these sensors less suitable
for longer exposure times. Furthermore,
state-of-the-art frontside-illuminated
(FSI) sensors can still be a better choice
in certain applications, showing excellent
performance also in low-light conditions
(Figure 2).
Beyond CCD and CMOS, a few ad-
ditional high-end sensor types are also
on the market, such as EMCCD (electron
multiplying CCD) and ICCD (intensified
CCD). However, these cameras are gener-
ally not considered for large products
today, as the technology is too expensive
and serves mostly niche applications.
CMOS technology will continue to
evolve in the years to come; fluorescence
microscopy can be expected to be domi- Figure 3. Because of red, green, and blue color filters on the pixels, the quantum efficiency is lower
nated by this state-of-the-art technology in a color camera compared to a monochrome camera. In addition, color cameras typically come
in the future, while further developments with an IR-cut filter blocking light of ~650 to 700 nm upward.

BioPhotonics • September 2019 33

919BI_FEAT Flourescence.indd 33 9/5/2019 8:14:20 AM

 Fluorescence Microscopy

monochrome cameras. Selectable light

sources and filter sets provide the right
combination of excitation and emission
wavelengths for each fluorophore used
(Figure 4).
However, certain applications may
create a demand to do color imaging and
fluorescence within one instrument using
only one camera. This is possible if the
sensitivity demands of the fluorescence
application are not too high.

Global and rolling shutters

CCD sensors have only one shutter
type (global) while CMOS sensors are
available in two types: rolling and global.
Choosing the right sensor has a signifi-
cant impact on image quality, especially
when target objects are moving. In rolling
shutter sensors, the pixels are exposed
line after line. As a result, an object that
has changed its position between signal
capturing of two lines produces deviating
image situations, generating space distor-
tion in the image. A technical advantage
of rolling shutter sensors is that they have
fewer electronic parts in the pixel, which
can result in less noise during readout.
Meanwhile, global shutter sensors expose
all pixels of the sensor at the same time.
In this case, there is no time shift between
the exposures of the different pixel lines,
thereby generating no space distortions
when objects are moving.
Sensitivity and dynamic range
Before taking a closer look at the qual-
ity of an image, it is important to ensure
Figure 4. Two fluorescence images of a commercially available BPEA (bovine pulmonary artery
endothelial) cell substrate, taken with a front-illuminated monochrome IMX174 CMOS sensor in a Basler
MED ace 2.3 MP mono camera (exposure times: 500 ms DAPI, 5 s Alexa Fluor 488, 63× magnification).
Both single images, showing different cell structures (cell nucleus and filamentous actin), are then
colored by software and merged, showing both structures in one image.
that the system is sensitive enough to The higher the QE, the better the yield of
capture the fluorescence signals, which photons, enabling shorter exposure times,
can be very weak, depending on the reducing photo bleaching of fluorophores,
individual application. Sensitivity should and potentially improving overall imag-
be understood as the minimum amount of ing speed.
light that is required to generate a signal Often it is also beneficial to have a
that can be distinguished from noise. An wide range of light intensities that can be
important value is the quantum efficiency resolved with one exposure. Here the full
(QE) describing the ratio between the well capacity is relevant. It describes the
incident photons of the light source and maximum number of electrons that can
the generated electrons of the pixel. It be generated by one pixel per exposure.
depends on the wavelength, and to get The higher the full well capacity, the
the best result, the spectrum of a given more light can be captured before a pixel
sensor should fit with the emission spectra is saturated, reducing the requirement of
of the fluorophores in the application. additional exposures due to saturation.

Figure 5. Comparison of the noise behavior (temporal dark noise) of Figure 6. Comparison of the noise behavior (with dark current noise) of
CCD and CMOS cameras with an exposure time of 10 ms. CCD and CMOS cameras with an exposure time of 4 s.

34 BioPhotonics • September 2019

919BI_FEAT Flourescence.indd 34 9/5/2019 8:14:23 AM

 Combining the maximum number
of electrons with the lowest number of
electrons required to produce a true signal
(see “read noise” in the next section), the
dynamic range characterizes a camera’s
overall ability to measure and distinguish
different levels of light.
Finally, there is the absolute sensitivity
threshold, which is the number of pho-
tons required by one pixel to generate a
signal-to-noise ratio (SNR) of 1 — mean-
ing the signal is equivalent to the noise.
The smaller this value, the less light is
required to produce a true signal. Because
it does not take into account the pixel size,
it cannot be directly used to compare two
cameras when their pixel sizes are differ-
ent.
Depending on the quantification
requirements of the application and
the technical dynamic range of a given
camera, the bit depth of the pixel deter-
mines the number of gray values that can
be differentiated. Typical machine vision
cameras have a depth of 8 bit (28 or 256
gray values), which is already sufficient
for visual needs, since the human eye can
BioPhotonics • September 2019
919BI_FEAT Flourescence.indd 35
only resolve 6 to 7 bit. Beyond that,
12 bit (212 or 4096 gray values) is common
and technically sufficient in the major-
ity of cases. However, certain scientific
applications demand 14 or even 16 bit
(214/216 or 16,384/65,536 gray values). It is
obvious that image file sizes significantly
increase with the bit depth, requiring
more IT resources for processing and
storing; interface bandwidth may also
create a bottleneck at even lower frame
rates. However, for the majority of cases,
the exposure time will probably be the
limiting factor that inhibits very fast sen-
sor readouts.
Image quality and noise
Noise is the deviation between the
true signal value and the value that is
produced by a measuring system. The
SNR quantifies the overall noise of an
imaging system at a certain light level
and is a common parameter used to
compare cameras. The higher the SNR,
the better the image quality. In the
imaging process, there are types of noise
that can only rarely — if at all — be
reduced by the camera technology (e.g.,
photon/shot noise, which is caused by the
photons’ physical appearance). However,
other noise types that influence image
quality are significantly affected by the
sensor itself and the camera technol-
ogy. In recent years, the former CCD
technology was surpassed in the areas
of image quality and performance by
modern CMOS sensors. Read noise — or
temporal dark noise — is the noise added
to a signal per one shutter event and is
given in eˉ/pixel. Modern CMOS sensors
go down to a read noise of only 2 eˉ/pixel
(Figure 5).
Another noise source that is relevant
for fluorescence applications becomes
important when exposure times increase;
it is caused by dark current. Dark current
is the leakage of electrons during expo-
sure and is expressed in eˉ/pixel/s (Figure
6). As a rule of thumb, the dark current
doubles with each temperature increase
of 7 °C. Noise types that describe not a
temporal- but a space-related behavior are
called fixed-pattern noise; this describes
deviations that can be seen between dif-
35
9/5/2019 8:14:26 AM
 Fluorescence Microscopy
Fluorescence in the Field
Fluorescence is a physical phenom-
enon and not just a concrete technol-
ogy. The possible methods — for
analytics, quantitative determinations,
or visualizations used in the life sci-
ences, for example — are virtually
infinite. Fluorophores can be coupled
to various carriers such as proteins
(often antibodies), nucleic acids, or
microparticles. But they can also be
integrated as gene technology markers
in organisms to examine cell-biological
functions and processes. The following
examples show the versatile applica-
tion options for fluorescence.
In the in vitro diagnosis of autoim-
mune or infectious diseases, one
can use the technique called indirect
immunofluorescence microscopy to
detect autoantibodies in the patient’s
blood.
In addition to manual microscopy,
there are already automated systems
that give lab physicians suggested
findings based on fluorescence pat-
terns of the cells incubated with pa-
ferent pixels. It can be caused by the pixel
electronics or by inconsistent tempera-
tures over the sensor area. Standardized
quantification measures of these noise
types are the DSNU (dark signal nonuni-
formity), which describes the deviation
of generated electrons without any light
signal, and the PRNU (photoresponse
nonuniformity), describing the pixel-to-
pixel deviation at a certain light level.
By setting cutoff values on pixel-to-pixel
deviations, one can further differentiate
and describe outlying pixels as defect
pixels, such as hot pixels, that show high
gray values without a corresponding
signal. Certain camera manufacturers
already correct defect pixels during qual-
ity control by interpolation of neighboring
pixels so integrators are not impaired by
these artifacts.
There are several different interfaces
on the market. To decide which interface
is required, one should look at the follow-
ing points, depending on the individual
application: data/image rate, cable length,
standardization, integration effort, and
costs. The interface technologies USB 3.0
(renamed 3.2 Gen 1) and GigE represent
36
919BI_FEAT Flourescence.indd 36
tient serums, as evaluated by software
(Figure 7). Another system, in turn,
analyzes patient serums on malaria
pathogens in less than 3 minutes. The
analysis is performed with vision-
based algorithms that also take fluo-
rescence signals into consideration.
Point-of-care systems are increas-
ingly gaining significance in medical
Figure 7. Computer-aided immunofluorescence
microscopy system EUROPattern (EUROIMMUN
Medizinische Labordiagnostika AG, Germany)
for autoimmunity and infection diagnostics using
Basler ace CMOS cameras for fluorescence
imaging.
the current state-of-the-art interfaces that
are feasible for integration into fluores-
cence microscopy-based systems. For
both interfaces, vision standards are
available that provide specifications de-
veloped by leading camera manufacturers
to improve vision system design, effort,
and performance for the camera integra-
tors.
USB 3.2 Gen 1 is the common and
established plug-and-play interface of-
fering the easiest possible integration,
enabling data rates of 380 MB/s (e.g.,
75 fps at 5 MP) — sufficient for the
majority of applications and with cable
lengths of up to a few meters, including
power supply, and also supporting mul-
ticamera integration. GigE is used when
longer cable lengths and a more precise
synchronization of multiple cameras is
required. The bandwidth of GigE is 3.8×
slower (100 MB/s) than that of USB 3.0,
but 5 GigE and 10 GigE are becoming
available in forms that enable higher
data rates of 500 MB/s and 1000 MB/s.
For USB 3.0, new versions with up to
fourfold higher bandwidth have already
been published as well. However, they
Figure 8. A manual immunofluorescence microscope
with a Basler MED ace camera.
have not found their way into series vision
products as yet.
Integration of new standards typically
requires new accessories or peripherals,
which adds a delay in availability that can
negatively affect time to market because
of the limited range of established prod-
ucts.
Cooling
The temperature of the sensor has a
central impact on dark current, which
decreases the SNR and image quality —
especially when light signals are weak
and longer exposure times are required.
Cooling of cameras is therefore an impor-
tant topic, but not necessarily mandatory
in fluorescence imaging. As cooling
measures significantly affect the total cost
of ownership, the majority of cameras are
only passively cooled, which is already
sufficient for applications with strong
fluorescence signals. Still, the design of
these cameras has an impact on the sensor
temperature.
Heat production should be avoided by
designing the operation with low power
consumption, and heat should be ef-
BioPhotonics • September 2019
9/5/2019 8:14:29 AM
 diagnostics. Among other things, they
make it possible to establish better
medical care even in economically
and infrastructurally weak regions,
thanks to simple and inexpensive
applications. Lab-on-a-chip technolo-
gies enable the processing of patient
samples on a small chip, without
requiring complex lab equipment.
Fluorescence applications can also
be found in the practice of medicine.
Figure 9. Immunofluorescence microscopic assay
in autoimmune diagnostics (left). A DNA micro-
array used in cancer and other disease research
(right).
Images courtesy of Basler AG.
ficiently transported to the outside via the
internal hardware design (by the camera
manufacturer) and through mounting
the camera to a further heat-dissipating
carrier (by the camera integrator). For ac-
tive cooling of the sensor, thermoelectric
(Peltier) elements are used, and normally
an integrated fan removes the heat that is
generated by the Peltier element. The fan
also helps to prevent condensation on or
freezing of camera parts when their tem-
peratures are below ambient. If vibrations
(which can arise from the fan) need to be
prevented in the system, some cameras
can even be water-cooled. The longer
the exposure time, the lower the cooling
needs to be. Due to these variabilities, it is
not possible to list concisely the condi-
tions of the applications at which cooling
measures are actually required. But for
many applications it can be assumed that
up to levels of several seconds, the noise
caused by the dark current of modern
high-quality CMOS sensors does not have
a significant negative influence.
Beyond the summarized hardware and
sensor specifications, cameras can pro-
vide firmware features that are designed
BioPhotonics • September 2019
919BI_FEAT Flourescence.indd 37
In surgical microscopy, for example,
the operating doctor is increasingly
supported by specific fluorescence
markings of blood vessels or tumor
tissues, allowing the doctor to oper-
ate with perfect precision using FGS
(fluorescence-guided surgery). Den-
tists can also offer faster and more
specific treatment, such as making
tooth areas affected by decay selec-
tively visible during the treatment. Last
but not least, fluorescence-microscope
applications are used in pathology to
examine tissues from patient biopsies
for possible diseases.
The life sciences offer a broad
range of fluorescence-based ap-
plications in which microscopic
examinations have a significant
share. Immunofluorescence micros-
copy enables specific detection of
particular proteins — for example,
to detect or clarify their localization
in cells and tissues or as markers for
beginning cell death, depending on
particular test conditions (Figure 8).
to improve image quality in low-light
conditions.
One example is the defect pixel correc-
tion. During the final testing procedures
at Basler AG, the camera is tested at dif-
ferent exposure times, and defect pixels
are located and saved inside the camera’s
cache. In operation mode, the values of
defect pixels can be interpolated by the
weighted sum of the neighbor pixels. This
helps improve image quality and SNR.
Another firmware feature is called
“long exposure mode.” In this mode, the
camera produces less heat during expo-
sure and thereby runs at a lower internal
sensor temperature, enabling a lower
noise level. The feature also compensates
for sensor glow. In conclusion, checking
the availability of firmware features in
the manufacturer’s documentation may be
helpful for finding the right fluorescence
camera.
The current CMOS sensor generations
make applications possible that were for-
merly impossible without the investment
of multiple thousands of euros in a CCD
camera. These new possibilities become
more important as fluorescence becomes
Nowadays, live cell imaging can also
be performed for longer time periods
on automatic systems.
Miniaturization and parallelization
to increase analysis numbers are es-
pecially significant in pharmaceutical
research, since a very high number of
samples are screened analytically in
the search for new active substances.
This is where microarrays and high-
content screening systems are used
(Figure 9). With automatic colony
counters, fluorescence markers can be
used in petri dishes to select success-
fully transfected cells to subsequently
pick a sample of the respective
colony. This means it is verified
whether particular genetic material
was actually transferred to the cell
as part of an experiment, and the
researchers can continue using this for
their research.
Aside from the life sciences,
fluorescence-based methods are also
used in other areas, such as material
analysis and forensics.
an increasingly used tool in the life sci-
ences, making structures and processes
visible. And because of the discontinu-
ation of this product group by Sony, the
dominant global supplier, manufacturers
in the medical and life sciences need to
replace the CCD cameras integrated in
their instruments. Providing excellent
performance at reasonable prices, un-
cooled cameras with CMOS sensors can
be the right choice.
Meet the author
Felix Asche, Ph.D., is product market manager
for the Basler AG MED ace camera series.
After studying biology, he completed his
doctorate at the Bernhard Nocht Institute for
Tropical Medicine; he then worked for a lead-
ing diagnostics manufacturer as product man-
ager in the area of clinical chemistry for the
D/A/CH (Germany, Austria, and Switzerland)
region. Before joining Basler in 2016, he was
a product manager in the area of automation
for seven years for a leading German global
manufacturer of medical lab diagnostics.
37
9/5/2019 8:14:32 AM
 TPLSM has the potential to improve outcomes for cancer patients undergoing surgery. Courtesy of iStock.com/STEEX.

Open-Source Photon Counting

Advances Biological Research
Fast and volumetric intravital imaging represents the most demanding scenario
in which photon counting is drastically improving the performance of existing systems.

BY PABLO BLINDER, LIOR GOLGHER, AND

HAGAI HAR-GIL, TEL AVIV UNIVERSITY

A
surgeon removing a malignant
tumor1, a security agent detecting
harmful molecules at a stadium2,
an autonomous car avoiding a collision3,
a weather satellite spotting hurricane
formations4, a neuroscientist tracking
neuronal activity deep within the brain5
— all of these situations involve quickly
identifying small optical signals buried
within a large noisy volume. Efforts
made by many to achieve this difficult
goal have led to a “convergent evolution”
toward a common solution: time-tagged
photon counting using sensitive single-
pixel photodetectors. Consequently, free
and open-source software recently de-
veloped for neuroscientific research may
find its way to assisting clinicians and
researchers in otherwise unrelated fields.
Functional imaging at the cellular level
in the living brain is commonly achieved
with two-photon laser-scanning micros-
copy (TPLSM), which enables research-
ers to face the challenges imposed by
scattering of turbid media6. For example,
using this technique, researchers are able
to record the activity of individual cells
deep inside the living murine cortex,
with minimal perturbation to the imaged
animal. Although TPLSM has become
the gold standard for imaging under these
challenging conditions, overcoming exist-
ing trade-offs inherent with this technique
will expand the scope of the scientific
questions that could be asked.
In particular, as a point-scanning
technique, TPLSM forces microscopists
to balance between imaging rate (pixel
dwell time) and the size of the field of

38 BioPhotonics • September 2019

919BI_FEATBiological.indd 38 9/5/2019 8:13:03 AM

 Figure 1. Illustration of the difference between digital photon counting and analog integration acquisition schemes. The raw output from the detector
(center) is either summed in consecutive time bins for the analog integration or thresholded during photon counting. Courtesy of the Blinder Laboratory.

view. If one wishes to acquire a large

number of frames every second — per-
haps to capture fast dynamic processes —
it is expected that all pixels in each frame
will be darker, as less photons per frame
reach the detector. This trade-off becomes
more severe as scientists continuously try
to image larger volumes of the brain in a
rapid fashion6.
This trade-off mandates constant im-
provement of the collection optics, as well
as the use of very sensitive and precise
detectors to increase the probability that
each photon that does arrive at the detec-
tor will be tallied successfully. Photomul-
tiplier tubes are the workhorse of TPLSM.
For each detected photon, the detector
emits a characteristic electrical current,
referred to as a single electron response
(SER). The SER pulse height is highly
variable7. The detector also emits noisy
baseline current, even when no photon is
detected.
The signal coming out of the detector
can be processed by two main methods.
The common scheme used in most mi-
croscopes is known as analog integration
(Figure 1, bottom), in which the continu-
ous signal is summed after a short period
of time. This value is used as the bright-
ness value corresponding to the pixel. Figure 2. Photon counting reduces noise artifacts. The left column shows the sum of three frames as
The more photons arriving at the sample, captured with a well-established analog system. The noise is clearly visible when observing the Fourier
transform of that image (bottom). Yet the same imaging conditions provide an improved result
the higher that sum will be. However,
when using the photon counting imaging modality (right column), as seen in the resulting Fourier
analog integration also attributes a higher transform. The images show the cortex of a mouse 200 µm below the surface, genetically encoded to
brightness to photons that have randomly express fluorescent proteins in its neurons. They were captured at 15 Hz and span 335 × 335 µm2.
caused a higher SER and sums them with Scale bar = 50 µm. Fourier transform images are log-scaled. Reprinted/adapted with permission from
baseline noise fluctuations. These factors Reference 5, The Optical Society.

degrade the quality of the resulting image.

Alternatively, the second acquisition
scheme is digital photon counting (Figure
1, top). Its main principle is to threshold
the incoming SER signal from the detec-

BioPhotonics • September 2019 39

919BI_FEATBiological.indd 39 9/5/2019 8:13:08 AM

 Biological Research
Figure 3. Intravital volumetric imaging of a living fruit fly, genetically encoded to express fluorescent
proteins in parts of its brain. The entirety of the volume, recorded at 73.4 volumes per second and
spanning 234 × 600 × 330 µm3 (a). Responses of the rectangular areas similarly colored in (a) to
an odor puff (b, c). Scale bars = 50 µm. Reprinted/adapted with permission from Reference 5,
The Optical Society.
tor, registering only pulses that cross
some preset value. In this manner, the
output of the detector is digitized, produc-
ing a “1” if and when a photon is detected.
Importantly, a photon counter gives equal
weight to each photon detection event,
thereby eliminating most of the SER-
associated variance.
Previous implementations of photon
counting acquisition schemes are not
as well suited for the increasingly rapid
imaging conditions described above,
because the electronics used for the
thresholding and counting of the digital
events suffer from issues such as “dead
time” after each detected event, and low
bandwidth. While suitable devices were
available in other fields of research,
including nuclear physics, the process
of adapting them to biological imaging
was cumbersome and unclear. This led to
photon counting in a rather limited scope
because it requires either having a high
degree of electrical expertise to set up and
use to its full extent8 or opting for rather
expensive commercial solutions.
Photon counting
Recently, a new study has paved
the way to a wider adoption of photon
counting by dramatically simplifying the
integration of state-of-the-art electronics
into standard two-photon microscopes.
This innovation is driven by a new,
open-source application called PySight,
which aims to bridge the technological
gap between complex hardware and the
researchers who need to operate it5.
Embedding photon counting into
existing imaging systems using PySight
requires minimal modification — mainly
40
919BI_FEATBiological.indd 40
rerouting the output of the detectors into
commercially available off-the-shelf
hardware. It also takes into account the
timing signals from beam scanning
elements used in the microscopes (e.g.,
galvanometric and resonant mirrors). The
provided software analyzes the collected
data sets. With these pieces in place,
the day-to-day operation of the micro-
scope remains largely unchanged, while
the data is now saved in a time-tagged
photon-counting mode8.
The resulting images better reflect
the activity of the imaged cells, likely
because of the elimination of most SER-
associated variance and because of sup-
pression of background (Figure 2), overall
yielding a more reliable representation
of the underlying process that generated
the collected photons. In other words,
areas in the field of view that should be
dark because of lack of emitters remain
completely black in the resulting images,
whereas the recorded brightness of the
interesting features better corresponds
to their actual brightness. This results in
a net gain for most, if not all, imaging
experiments.
Furthermore, this unique data acquisi-
tion scheme allows labs to integrate ad-
vanced scanning elements almost seam-
lessly. This is because the position of the
beam steering elements is represented by
a simple synchronization signal recorded
along with the photon detection events.
The study’s researchers used the fastest
depth-scanning variofocal lens currently
available to obtain volumetric images,
rather than the typical 2D images gener-
ated by other TPLSM setups5 (Figure 3).
Previously, integrating this lens required
designing custom electronic circuits — a
job many labs do not wish to undertake.
Using PySight, however, integrat-
ing this advanced lens to achieve rapid
volumetric data is merely a single cable
connection away. The recorded volumet-
ric movie successfully captures rapid
changes in calcium concentration at
distinct areas of a fly’s brain as a response
to the presentation of different odors.
Aside from planar and volumetric im-
aging, time-tagged photon counting can
also be used for other imaging modalities
common in the field of microscopy, that
is, if they rely on bucket detectors such
as photomultiplier tubes. For example,
fluorescence lifetime imaging is the
practice of using information about the
decay rate of emitters to observe changes
in their state8. The new hardware and
software solution allows researchers to
collect data that can be used simultane-
ously for both modalities: fast, functional
and fluorescence lifetime imaging. It is
also extensible to wide-field compres-
sive multiphoton imaging9, which is an
emerging, promising technique for rapid
bioimaging at increased depths with
reduced phototoxicity.
Future applications
The applications of time-tagged photon
counting are not limited to neuroscience,
nor to basic scientific research. Nonlinear
microscopy is steadily migrating into
the clinical setting, already proving its
potential for improved tumor resection in
human patients1, as well as for label-free
tumor diagnosis, which may eventually
obviate biopsies.
A promising clinical future applica-
tion is two-photon photodynamic therapy,
namely, targeted destruction of cancer
cells using reactive oxygen species emit-
ted by photosensitizers that are excited by
NIR light10. By using time-tagged photon
counting to process multiphoton volu-
metric movies, surgeons could quickly
detect and target malignant cells lurking
hundreds of microns beneath unresected
human tissue, thereby potentially improv-
ing surgical outcomes in cancer patients.
While conventional analog integration
incurs writing to disk the sampled voltage
for each voxel, time-tagging photon-
counting solutions only write to disk a
sparse list of photon detection times.
Consequently, the size of the recorded
data sets only scales with the number of
detected photons, whereas the size of the
BioPhotonics • September 2019
9/5/2019 8:13:10 AM
 recorded data sets using analog integration grows with the num-
ber and size of the scanned dimensions. Time-tagged photon-
counting systems bear further potential for measuring the time
of flight of photons from a pulsed emitter to targets of interest,
thereby estimating their distance.
Such light detection and ranging (lidar) systems are used for
diverse applications, ranging from planetary mapping through
tracking of atmospheric cloud formation4 to obstacle detection
for self-driving cars3. Additionally, rapid volumetric hyperspec-
tral imaging may enable the detection of explosives and toxic
chemicals localized within a large venue2.
This diverse and growing community of time-tagged photon-
counting users has been enriched by use of free and open-source
tools such as PySight.

Meet the authors

Pablo Blinder, Ph.D., leads a multidisciplinary laboratory in the Neuro-
biology, Biochemistry, and Biophysics School’s life science faculty at
Tel Aviv University in Israel. He received a degree in neuroscience from
Ben-Gurion University and trained as a postdoctoral researcher in the
physics department at the University of California, San Diego.
Lior Golgher is a doctoral student at the Sagol School of Neuroscience
at Tel Aviv University. He has bachelor’s degrees in physics and electri-
cal engineering from the Technion Israel Institute of Technology.
Hagai Har-Gil is a graduate student at Tel Aviv University. He has a
bachelor’s degree in physics and is currently researching superresolution
methods at Tel Aviv University’s Sagol School of Neuroscience.

References
1. S.R. Kantelhardt et al. (2016). In vivo multiphoton tomography and
fluorescence lifetime imaging of human brain tumor tissue. J Neu-
rooncol, Vol. 127, Issue 3, pp. 473-482.
2. A.C. Geiger et al. (2019). Sparse-sampling methods for hyperspectral
infrared microscopy. Proc SPIE, Image Sensing Technologies: Mate-
rials, Devices, Systems, and Applications VI, Vol. 10980, p. 1098016.
3. F. Zhang et al. (2017). Adaptive strategy for CPPM single-photon
collision avoidance LIDAR against dynamic crosstalk. Opt Express,
Vol. 25, Issue 11, pp. 12237-12250.
4. T. Markus et al. (2017). The ice, cloud, and land elevation satellite-2
(ICESat-2): science requirements, concept, and implementation.
Remote Sens Environ, Vol. 190, pp. 260-273.
5. H. Har-Gil et al. (2018). Pysight: plug and play photon counting for
fast continuous volumetric intravital microscopy. Optica, Vol. 5,
Issue 9, pp. 1104-1112.
6. N. Ji et al. (2016). Technologies for imaging neural activity in large
volumes. Nat Neurosci, Vol. 19, pp. 1154-1164.
7. S. Moon et al. (2008). Analog single-photon counter for high-speed
scanning microscopy. Opt Express, Vol. 16, Issue 18, pp. 13990-
14003.
8. W. Becker, ed. (2015). Advanced Time-Correlated Single Photon
Counting Applications. Springer Series in Chemical Physics,
Vol. 111.
9. M. Alemohammad et al. (2018). Widefield compressive multiphoton
microscopy. Opt Lett, Vol. 43, Issue 12, pp. 2989-2992.
10. S. Wang et al. (2019). Polymerization-enhanced two-photon
photosensitization for precise photodynamic therapy. ACS Nano,
Vol. 13, Issue 3, pp. 3095-3105.

BioPhotonics • September 2019 41

919BI_FEATBiological.indd 41 9/5/2019 8:13:12 AM

 Neuroscience 2019 will take place at the McCormick Place Convention Center in Chicago Oct. 19-23. Courtesy of iStock.com/benkrut.
Neuroscience 2019 Celebrates
SfN’s 50th Anniversary
T
his fall, scientists and researchers
from around the world will gather
at the Neuroscience 2019 confer-
ence to hear lectures such as “Wavefront
Engineering: Illuminating the Neural
Landscape,” from Valentina Emiliani of
the Vision Institute in Paris. In her Presi-
dential Special Lecture, Emiliani will
discuss research related to a revolution in
optogenetics: the shaping of wavefronts to
guide light through tissues with millisec-
ond precision and single-cell resolution,
which is bringing the field into a new
phase called “circuit optogenetics.”
Optical technology is one subject of
the world’s largest scientific meeting,
Neuroscience 2019, which this year marks
the 50th anniversary of the founding of
the Society for Neuroscience (SfN) in
1969. The event will include a variety of
workshops, networking, and socializing
among peers.
42
919BI_NeuroscienceShow.indd 42
To be held Oct. 19-23 at the McCor-
mick Place Convention Center in Chi-
cago, the conference will commemorate
advancements in global neuroscience
research and also SfN’s mission of
advancing and advocating for scientific
exchange, supporting diversity and career
training, and educating the public.
Nearly 30,000 members of the scientific
community will gather for the event
and over 500 exhibitors will attend.
Visitors will learn about cutting-edge
technologies, covering topic areas such as
circuit development, neurodegenerative
disorders, the first FDA-approved drug
for spinal muscular atrophy, and more.
Attendees will learn from top research-
ers in the field, collaborate with other
professionals, and even earn continu-
ing medical education (CME) credits at
CME-designated lectures and symposia
during the conference.
To mark the 50th anniversary, Susana
Martinez-Conde will chair a storytelling
session titled “The Storytelling Brain:
How Neuroscience Stories Help Bridge
the Gap Between Research and Society.”
Neuroscience 2019 will host more than
50 symposia and minisymposia. Panel
session topics include adult hippocampal
neurogenesis, vision restoration, sensory
circuits, and artificial intelligence and
how it can affect neuroscience.
The conference will host four Presi-
dential Special lectures, five featured
Lectures, and 15 special lectures by
innovative neuroscientists and leading
researchers in the field.
In addition to Emiliani’s talk, there
will be three other Presidential Special
Lectures:
• “From Base Pairs to Bedside:
Anti-sense Modulators of RNA Splic-
ing to Treat Neurological Diseases,” by
BioPhotonics • September 2019
9/5/2019 8:31:23 AM
 Adrian R. Krainer of Cold Spring Harbor
Laboratory.
• “Understanding Cortical Develop-
ment and Disease: From Embryos to
Brain Organoids,” by Paola Arlotta of
Harvard University.
• “The Cell Biology of the Synapse and
Behavior,” by Daniel A. Colón-Ramos of
Yale University School of Medicine.
Other featured lectures will cover
topics such as dialogue between neurosci-
ence and society, the circadian clock, and
neuroethics. The featured History of Neu-
roscience lecture presented by Reinhard
Jahn of the Max Planck Institute for Bio-
physical Chemistry, titled “Exocytosis of
Synaptic Vesicles: From Quantal Release
to Molecular Machines,” will give a his-
tory of synaptic vesicles and how research
has advanced since their discovery in the
1950s. Neuroscience 2019 will host over 50 symposia and minisymposia on a wide range of topics. Courtesy of
Lecture topics are categorized into Joe Shymanski/Society for Neuroscience.
nine main themes: development; neural
excitability, synapses, and glia; neuro-
degenerative disorders and injury; sen-
sory systems; motor systems; integrative
physiology and behavior; motivation
and emotion; cognition; and techniques.
Lectures will include “Evolution and
Dissolution of Memories Over Time” by
Eleanor A. Maguire of University Col-
lege London, and “Neural Mechanisms
of Short-Term Memory and Motor Plan-
ning” by Karel Svoboda of the How-
ard Hughes Medical Institute, Janelia
Research Campus.
Neuroscience 2019 will host three
roundtable panel discussions to encour-
age debate between clinician-scientists on
topics such as exoskeletons and robotics
for neurorehabilitation, gene therapy, and
the mechanisms of drug addiction.
For those who wish to learn from ex-
perts before the official start of the meet-
ing, the SfN Pre-Conference Sessions
will feature short courses and “Meet the
Experts” sessions that will discuss emerg- Nearly 30,000 members of the scientific community and over 500 exhibitors will gather for Neuroscience
2019. Courtesy of Joe Shymanski/Society for Neuroscience.
ing topics and techniques in scientific
research.
Also at Neuroscience 2019: poster
sessions; SfN-sponsored socials such as The extensive exhibition will host over
the International Brain Bee Social, the 500 booths and companies showcasing For more information about
Neuroethics Social, and the Music Social; the latest products and relevant, trailblaz- Neuroscience 2019, visit
dozens of satellite events; professional ing technologies. Some of the many com- www.sfn.org/Meetings/
development events such as the Gradu- panies planning to attend include Thermo Neuroscience-2019.
ate School Fair and NeuroJobs Career Fisher Scientific, BioTek Instruments,
Center; and workshops including “How Andor Technology, MKS Instruments,
to Thrive as a Woman in Neuroscience” and ThorLabs.
and “Bringing Genetic Diversity to Model
Organism Research.”

BioPhotonics • September 2019 43

919BI_NeuroscienceShow.indd 43 9/5/2019 8:31:27 AM

 Q&A
Spectroscopy gives clear view of data
Spectroscopy is a broad field comprising a variety of methods and modalities, and cover-
ing applications from industry process control and security to biomedical. Each modality
has its particular advantages and disadvantages, including speed, sensitivity, and ease of
implementation. Nevertheless, they share common factors and trends.
Photonics Media spoke with Iwan W. Schie, leader of the Multimodal Instrumenta-
tion work group in the Leibniz Institute of Photonic Technology’s (IPHT’s) Department
of Spectroscopy and Imaging in Germany. He has a doctorate in biomedical engineering
from the University of California, Davis. During his time in the program, his research
focused on instrument development and medical applications for multiphoton microscopy
and spontaneous Raman spectroscopy. In his current position with Leibniz-IPHT, he is
researching high-throughput Raman spectroscopy systems for single-cell classification,
multimodal fiber-probe development, and instrumentation for medical in vivo applications.
We asked him to share his take on recent advancements in spectroscopy, exciting areas
of research, and what the future holds for the field.

Q: What trends are you seeing

in the spectroscopy field?
On the instrumentation side spe-
cifically, the trend is the development of
Both come at a significant time cost,
making it challenging for the casual user

A: From the research side, the most

common trend for spectroscopic modali-
ties is the combining of methods, most
frequently with optical modalities, to
increase the wealth of information about
a sample. Some spectroscopy modalities,
such as Raman spectroscopy, tend to be
slow but have a spectacular informational
depth. For example, in the medical field
it is quite advantageous to combine Ra-
man spectroscopy with methods such as
optical coherence tomography (OCT),
fluorescence lifetime imaging microscopy
(FLIM), or even fluorescence spectros-
copy. Also advantageous is the use of
faster methods as a red-flag technology
for the identification of regions of interest,
and to use Raman spectroscopy to provide
a precise molecular profile, which can be
used for diagnostics.
Obvious general trends, including the
application of artificial intelligence (AI)
approaches for spectroscopic data evalu-
ation, are also more frequently found
in the field. However, here the spec-
troscopy community has already been
at the forefront of machine-learning
approaches (frequently termed chemo-
metrics) which were applied for decades
to better comprehend spectroscopic data,
and which in most situations have a
multivariate structure. As such, broader
availability of larger data sizes will en-
able a more comprehensive use of these
approaches.
compact and high-performance spectrom-
eters and detectors, which results in an
upswing for spectroscopic applications in
the consumer market. Today’s consumers
are more data-driven, and have sharp-
ened awareness of their habits, using a
variety of wearable tracking devices to
monitor sleep patterns, calorie intake,
or pulse. Here, spectroscopic modalities
offer new ways for monitoring — for
example, chemically evaluating the fresh-
ness of dietary products or determining
authenticity of produce. Who would not
like to have a mobile phone that provides
chemical information about their dietary
products or lets them chemically identify
things in their surroundings?
Q: What exciting projects
are you currently working on?
A: In particular, molecular spectros-
copy offers the possibility to identify
and characterize chemical signatures of
samples rapidly, nondestructively, and
without any preparation steps. At least
this is the premise for using molecular-
sensitive spectroscopy methods such as
vibrational spectroscopy, especially in the
field of biomedical applications. However,
when looking at current devices and com-
monly performed applications, current
spectroscopy devices heavily require hu-
man interaction, whether to perform the
experiments or to evaluate the data.
who is simply interested in applying the
method to get clear-cut information. To
overcome this, one of our own specific re-
search topics involves developing devices
— or an entire application procedure for
that matter — that require little to no
user interaction, harvesting all of the
advantages of spectroscopic approaches
but providing instantaneous and easy-to-
understand answers for the user. While
this sounds easy, it requires a comprehen-
sive understanding of all of the different
aspects of spectroscopy — instrumenta-
tion, data processing, and sample han-
dling — to achieve this goal.
On par with this topic is the develop-
ment of high-throughput Raman devices
for a large-scale sampling of cells. Here
again, the proper device development
and application to real-world scenarios
significantly increases the attraction
of the method. Another topic related to
device translation to clinical applica-
tions is that it requires not only extensive
engineering know-how but also has
to consider the regulatory framework
— MDR 2017/745 — which imposes
extensive regulatory rules for moving
research devices to real in vivo medical
applications.
From the point of view of a research
institute, this is a particularly challeng-
ing and comprehensive undertaking. The
combination of spectroscopic modalities,
as described above, is also of significant
interest.

44 BioPhotonics • September 2019

919BI_Q&A.indd 44 9/5/2019 8:29:12 AM

 Q: What implications for the
industry are emerging from spectros-
copy research and technology?
process and provide instant information.
Quite frequently, large batches have
to be discarded, resulting in significant
number of feasible applications shown.
However, it is challenging to have studies
with hundreds of patients and in differ-

A: Spectroscopy applications are

gaining significant interest in industrial
process controlling because they provide
immediate information about chemi-
cal signatures, concentration profi les,
modifications of chemical structures, and
changes in chemical ratios. Each set of
accessible information is an important
factor for process controlling.
Processes are usually analyzed by
random sampling of the product, which
has to be sent for extensive analysis to
external laboratories. This is not only
time-consuming but also a significant cost
factor. The reader would be surprised to
know how many production processes are
controlled by the experience of the user or
controlling parameters, which have been
established empirically and not controlled
through closely monitored, in-line analyt-
ical tools. Spectroscopy-based methods,
however, can nondisruptively profi le the
economic damage to the companies and
possible damage to the environment.
Here, the spectroscopy-based methods
could have a significant impact on differ-
ent producing industries.
Q: What does the future hold
for spectroscopy (in R&D,
diagnostics, etc.)?
A: Spectroscopy is currently on the
brink of a transformation, primarily
driven by the development of sophisticated
devices and improved components at re-
duced cost. This, combined with miniatur-
ization and a general desire for an increase
of monitoring and access to information in
consumer electronics, will provide signifi-
cant drive and further increase accessibil-
ity and novel applications.
In terms of medical applications in
diagnostics, there has been an increasing
ent clinical settings to properly validate
modalities and to further establish the
acceptability in the medical field.
This problem of acceptability is not
unique to spectroscopy; it is a challenge
for many other optical methods. For
industry applications, it is much easier
to translate spectroscopy because the
regulatory framework and experimental
evidence are much easier to attain than
in the medical field. Here, actually, the
developer of particular industrial equip-
ment can push new in-line and on-line
analytical modalities and implement them
in their equipment, which will be more
acceptable by the customers.
The last decade has seen spectacular
developments in spectroscopic applica-
tions, and in the next decade there will
be even more possibilities. Overall, the
next decade will see new excitement in
spectroscopy.

Optical Biomedical Imaging

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330 pages, 48 articles
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 APPOINTMENTS
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 ADVERTISERINDEX
Photonics Media Advertising Contacts
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 POSTSCRIPTS
The faster the flow, the brighter the glow
G
oing with the flow is rarely a bad thing. Unless you’re
talking about the notorious pathogen Pseudomonas.
The bacterium is almost always surrounded by flow-
ing water, and researchers are using part of that phenomenon to
measure the flow of cells within the body.
They discovered that the pathogen detects flow and changes
its behavior accordingly by activating genes that the Princeton-
based researchers have dubbed “fro,” for “flow regulated
operon.” They then used gene cloning to engineer a connection
between fro and a yellow fluorescent protein that causes the
bacterium to glow, said Joseph Sanfilippo, a lead author on the
paper.
“Fro’s response is not just an on-off switch; it’s actually tuned
to the speed,” Sanfilippo said. “It’s more like a dimmer switch
than a light switch.”
The researchers viewed the bioluminescent response under a
microscope, and what they saw was a real-time, visual speedom-
eter. At slow shear rates (the rate at which adjacent layers of fluid
pass by one another), the bioluminescence was dim. But at faster
rates, the bacterium shone brightly.
What they perhaps didn’t expect was a significant revelation
about the way the scientific community understands the biologi-
cal perception of flow.
A team of Princeton University biologists and engineers led by Zemer Gitai bioengineered a real-time bacteria speedometer by linking a flow-detecting gene
in Pseudomonas aeruginosa to one for illumination: The faster the flow, the brighter the glow. They discovered that flow detection occurs independent of force,
prompting new questions about how bacteria sense their environments. Courtesy of Matilda Luk/Princeton University Office of Communications.
50
919BI_Postscripts.indd 50
“Other researchers have found that different bacteria can
respond to fluid flow, and they’ve effectively assumed that it
[sensed] the force,” said professor Zemer Gitai, senior author on
the paper. “The intuition was so strong that [the stimulus] should
be force that, in fact, people didn’t bother to test this.”
Through experiments with fluids of varying viscosities, the
researchers found that fro responded to shear rates between 40
and 400 per second. To put that into perspective, blood within an
average-sized human vein flows at a shear rate of about 100 per
second.
“The speeds that fro responds to are the speeds that are going
through your body right now,” said Sanfilippo.
The revelation that some bacteria are able to measure and
react to flow may open up new avenues for antibiotic treatments,
especially for blood infections such as sepsis, said Joanne Engel,
chief of infectious disease and professor of microbiology and
immunology at the University of California, San Francisco.
The research was published in Nature Microbiology (www
.nature.com/articles/s41564-019-0455-0).
Joel Williams
joel.williams@photonics.com
BioPhotonics • September 2019
9/5/2019 8:28:27 AM
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