Fluorescence Microscopy: Super-Resolution and other Novel Techniques

Fluorescence Microscopy

Super-Resolution and other Novel Techniques

Editors

Anda Cornea

P. Michael Conn

Table of Contents

Cover image

Title page

Copyright

Preface

List of Contributors

Chapter 1. Evanescent Excitation and Emission

Introduction

Evanescence in General

Evanescence in Excitation: TIR

Evanescence in Emission: The Fluorophore Near Field

Evanescence from a Point Source: NSOM

Chapter 2. Adaptive Optics for Fluorescence Microscopy

Introduction

Aberrations in Microscopy

Principles of Adaptive Optics

Adaptive Aberration Correction in Fluorescence Microscopes

Wavefront Sensing

Schemes for Modal Sensorless Adaptive Optics

Conclusion

Chapter 3. Rapid Measurements of Orientation and Rotation in Ex Vivo Muscle Made Possible by Studying Small Number of Cross-Bridges

Introduction

Kinetics

Distribution of Cross-Bridge Orientations

Conclusions

Chapter 4. High Spatiotemporal Bioimaging Techniques to Study the Plasma Membrane Nanoscale Organization

Introduction

Spatial Resolution

Temporal Resolution

Conclusions and Outlook

Chapter 5. High-Resolution 3D Imaging of Intact Transparent Organs by 3DISCO

Introduction

Current Technologies for Imaging the Intact Brain

3DISCO

Chapter 6. Using RNA Mimics of GFP to Image RNA Dynamics in Mammalian Cells

Introduction

Studying Trinucleotide-Repeat-Containing RNA

Optimizing Imaging of CGG Repeat-Containing RNA

Time-Lapse Imaging of CGG-Repeat-Containing RNAs

Monitoring CGG Repeat Foci During Cell Division

Examining the Stability of CGG60 RNA

Imaging the Effects of Small-Molecule Drugs on CGG Foci

Imaging Other RNAs Using Spinach2

Troubleshooting Imaging with Spinach2-Tagged Constructs

Imaging Rare RNAs

Future Directions

Chapter 7. Surface Enhanced Raman Scattering (SERS) Image Cytometry for High-Content Screening

High-Content Screening: Status and Needs

Labels and Probes for HCS: Fluorescence and SERS

Spectral Imaging for Fluorescence and SERS

SERS Imaging in Cell-Based Assays

Status and Prospects

Chapter 8. Light Sheet Fluorescence Microscopy Applications for Multicellular Systems

Introduction

LSFM Implementations: A Universal Concept with Many Acronyms

LSFM at the Macroscopic Scale

3D Cancer Imaging Model: MCTS

Calcium Imaging, Fast Volumetric Calcium Imaging

Conclusions

Chapter 9. Correlative Light Electron Microscopy as a Navigating Tool for Cryo-Electron Tomography Analysis

Introduction

Electron Microscopy in Cellular and Structural Biology

Sample Preparation for Electron Microscopy

Cryo-Electron Tomography

Correlative Light and Electron Microscopy (CLEM)

Correlated Light and Electron Microscopy Direct Observation in Cryogenic Temperatures

CLEM Prospects

Chapter 10. Cellular and Molecular Applications of Super-resolution Microscopy

Introduction

Super-resolution Microscopy Techniques

Troubleshooting Sample Preparation

Thick, Living, Multicolor 3D Samples: Friends and Foes of Super-resolution Microscopy

Biological Applications

Conclusions

Chapter 11. Use of Engineered Nanoparticle-Based Fluorescence Methods for Live-Cell Phenomena

Introduction

Materials and Methods

Results and Discussion

Conclusions

Perspectives

Chapter 12. High-Resolution Estimation of Multiple Cell Populations in Tissue Using Confocal Stereology

Introduction

Design-Based Stereology as a Quantitative Tool

Sampling Requirements to Achieve a Population Estimate with High Resolution

Practical Requirements for Performing Stereology

Separation of Image Acquisition and Probe Analysis

Implementation of Systematic Random Sampling on a Confocal Microscope

Virtual Tissue with Post-Acquire Sampling

Conclusions

Conflict of Interest Disclosure

Chapter 13. Multiphoton Microscopy Applications in Biology

Introduction

Multiphoton Microscopy Applications

Chapter 14. Super-resolution Microscopy: A Comparison of Commercially Available Options

Introduction

Technical Considerations

Single-Molecule Localization Microscopy

Structured Illumination Microscopy

Stimulated Emission Depletion Microscopy

Summary

Future Directions and Emerging Techniques

Chapter 15. Structured Illumination Microscopy

Introduction

Theory

Materials and Methods

Results

Discussion

Chapter 16. The Role of Image Analysis Algorithms in Super-resolution Localization Microscopy

Introduction

Localization Algorithms and the Problem of Noise

Guidelines for Selecting a Localization Algorithm

Dipoles and 3D Localization

Rejection Algorithms for Multi-Molecule Images

Rejection Issues in 3D

Speeding Up Performance with Multi-Molecule Analysis

Conclusions

Index

Copyright

The cover shows 3D reconstruction of neuronal networks in the unsectioned mouse spinal cord obtained by 3DISCO method. The images courtesy of Dr. Ali Ertürk. Top image originally published in Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Ali Ertürk, Christoph P Mauch, Farida Hellal, Friedrich Förstner, Tara Keck, Klaus Becker. Nature Medicine 13:1 (166-172) Nature Publishing Group, January 2012.

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Preface

Microscopy has long been a cornerstone in our knowledge of the living world, but the last decade has seen tremendous advances that challenged earlier rules and limitations. Optical imaging has evolved into a valuable, albeit complex, analytical and quantitative tool. At one end of the size spectrum, the theoretical limit of optical resolution was broken, bridging the gap to electron microscopy. At the other end, whole organs and even organisms may now be imaged at the cellular level, revealing a three-dimensional architecture that was difficult to grasp in traditional two-dimensional sections. Progress in micromechanics, opto-electronics, computer power, molecular biology, and chemistry has allowed advanced concepts in physics to provide commonly used technologies in the service of biomedical sciences.

This volume reviews some of the most influential recent advances in fluorescence microscopy with an emphasis on super-resolution techniques, light-sheet microscopy, sample preparation, new labels, and analysis techniques.

Authors were selected based on their research contributions in the area about which they have written and on their ability to describe their methodological contributions in a clear and reproducible way. They have been encouraged to make use of graphics and comparisons to other methods and to provide tricks and approaches not revealed in prior publications that make it possible to adapt methods to other systems. In some cases, the reader will see helpful video to guide their work.

The editors want to express appreciation to the contributors for providing their contributions in a timely fashion and to the editors and staff at Academic Press for helpful input.

List of Contributors

Daniel Axelrod,     Departments of Physics, Biophysics, and Pharmacology, University of Michigan, Ann Arbor, MI, USA

Melanie Bokstad,     Ben Gurion University, Be’er Sheva, Israel

Martin J. Booth,     Department of Engineering Science and Centre for Neural Circuits and Behaviour, University of Oxford, United Kingdom

Julian Borejdo,     University of North Texas, Health Science Center, Fort Worth, TX, USA

Alessandra Cambi,     Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

Julien Colombelli,     Institute for Research in Biomedicine, Barcelona, Spain

Ali Ertürk,     Genentech, Inc., San Francisco, CA, USA

Eugenio F. Fornasiero,     European Neuroscience Institute and University of Göttingen, Göttingen, Germany

Sung-Sik Han,     Department of Life Science, Korea University, Seoul, Republic of Korea

Samie R. Jaffrey,     Weill Medical College, Cornell University, New York, NY, USA

Min-Kyo Jung,     Department of Life Science, Korea University, Seoul, Republic of Korea

Sandra de Keijzer,     Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

Jun-Sung Kim,     BITERIALS Co., Ltd., Seoul, Republic of Korea

Lynlee L. Lin,     Translational Research Institute, Woolloongabba, Queensland, Australia

Er Liu,     La Jolla Bioengineering Institute, San Diego, CA, USA

Corinne Lorenzo,     University of Toulouse and Centre National de la Recherche Scientifique, Toulouse, France

Ohad Medalia,     Ben Gurion University, Be’er Sheva, Israel; University of Zurich, Zurich, Switzerland

Marjolein B.M. Meddens,     Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

John P. Nolan,     La Jolla Bioengineering Institute, San Diego, CA, USA

Felipe Opazo,     European Neuroscience Institute and University of Göttingen, Göttingen, Germany

Chan-Gi Pack,     RIKEN, Wako, Japan

Brian R. Patton,     Department of Engineering Science and Centre for Neural Circuits and Behaviour, University of Oxford, United Kingdom

Daniel A. Peterson,     Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA

Jeremy Pike,     The University of Birmingham, Birmingham, United Kingdom

Tarl W. Prow,     Translational Research Institute, Woolloongabba, Queensland, Australia

Joshua Z. Rappoport,     The University of Birmingham, Birmingham, United Kingdom

E. Hesper Rego,     Harvard School of Public Health, Boston, MA, USA

Yasushi Sako,     RIKEN, Wako, Japan

Lin Shao,     Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

Alex Small,     California State Polytechnic University, Pomona, CA, USA

Mi-Roung Song,     BITERIALS Co., Ltd., Seoul, Republic of Korea

Shane Stahlheber,     California State Polytechnic University, Pomona, CA, USA

Rita L. Strack,     Weill Medical College, Cornell University, New York, NY, USA

Jennifer A. Thorley,     The University of Birmingham, Birmingham, United Kingdom

Miko Yamada,     Translational Research Institute, Woolloongabba, Queensland, Australia

Chapter 1

Evanescent Excitation and Emission

Daniel Axelrod     Departments of Physics, Biophysics, and Pharmacology, University of Michigan, Ann Arbor, Michigan, USA

Abstract

Evanescent light—light that does not propagate but instead decays in intensity over a subwavelength distance—plays a role in fluorescence microscopy in both excitation (i.e., total internal reflection, or TIR) and emission (i.e., supercritical angle fluorescence). This chapter describes the physical connection between these two forms as a consequence of geometrical compression of wavefront spacing and describes newly established or speculative applications and combinations of the two. In particular, each form can be used in analogous ways to produce surface-selective images, to examine the thickness and refractive index of films (such as lipid multilayers or protein layers) on solid supports, and to measure the absolute distance of a fluorophore to a surface. In combination, the two forms can further increase selectivity and reduce background scattering in surface images. The polarization properties of each lead to more sensitive and accurate measures of fluorophore orientation and membrane micromorphology. The phase properties of evanescent excitation lead to methods of creating a submicroscopic area of TIR illumination or enhanced-resolution structured illumination. Analogously, the phase properties of evanescent emission lead to a method of producing a smaller point spread function, in a technique called virtual supercritical angle fluorescence. This chapter emphasizes the concepts and theory (rather than experimental protocols and results) of evanescence for both excitation and emission, as well as the theory of its many existing and possible future applications.

Keywords

microscope imaging; near field; point spread function; polarization; supercritical angle; super-resolution; total internal reflection

Introduction

Some of the various super-resolution microscopy techniques share a common feature in that they attempt to exceed the standard light microscope resolution limit by employing evanescent light that decays in at least one direction in a distance much shorter than the wavelength. This group includes total internal reflection fluorescence microscopy (TIRFM; covered in another chapter in this book), near-field scanning optical microscopy (NSOM), and virtual supercritical angle fluorescence (vSAF) microscopy.¹–⁴ In some cases, evanescence is in the excitation light, in other cases it is in the emission, and in some it is in both. This chapter explores the physical concepts that these techniques share and points toward some established and more speculative possible directions for future work in evanescence-based super-resolution. The effect of evanescence on the use and detection of polarization, in both excitation and emission, is covered in considerable detail.

Evanescence in both excitation and emission can be understood as a response to geometrical compression, or squeezing, of wavefront spacing in at least one dimension. Evanescent light can be converted to or from propagating light traveling at supercritical angles relative to a nearby interface. Evanescence has numerous applications in fluorescence microscopy. Here is a preview of those to be discussed in this chapter:

• On the excitation side

• Supercritical excitation (TIRF) is commonly used for selective excitation of surface-proximal molecules, cell/substrate contact regions, and membrane-proximal cytoplasmic organelles.

• Variable angle TIRF has been used to deduce the concentration of fluorophores as a function of distance from the substrate.

• TIRF intensity vs. incidence angle on film-coated surfaces can display a resonance behavior that may measure the thickness, refractive index and possible lateral heterogeneities of surface-supported multilayer lipid or protein coatings.

• TIRF on film-coated surfaces can enhance the evanescent intensity by at least an order of magnitude.

• Polarized excitation TIRF can highlight submicroscopic irregularities in the plasma membrane of living cells and orientation of single molecules.

• Intersecting TIRF beams can extend the super-resolution of structured illumination.

• Radially polarized ring TIR illumination at the back focal plane (BFP) can produce a uniquely small illumination volume, possibly useful for fluorescence correlation spectroscopy and high-resolution scanning.

• The evanescent field at an NSOM tip facilitates the mapping of distances to fluorophores and surface topology.

• On the emission side

• The emission intensity pattern in the supercritical zone of the BFP reports the fluorophore concentration profile as a function of distance to the surface.

• The ratio of emission power in the supercritical vs. subcritical BFP zone can sensitively report absolute distance of a fluorophore to the surface to an accuracy of tens of nanometers.

• Taking into account the interaction of the fluorophore near field with a surface alters the predicted depolarization induced by high-aperture observation.

• For a film-coated surface (such as a lipid multilayer), the emission intensity pattern in the supercritical zone of the BFP is uniquely sensitive to film thickness.

• On both excitation and emission sides

• By combining the vSAF emission image protocol with standard TIRF excitation, an even higher degree of surface selectivity should be attainable than from either individually, with much less scattering background intensity.

Evanescence in General

For propagating light, the shortest spacing (λm) between each traveling wavefront (i.e., the periodic locus of points of equal phase) for propagating light is simply given as λm = λo/nm, where λo is the light wavelength in vacuum, and nm is the refractive index of medium m (m = 1, 2, 3 will be used here for a system of stratified planar layers). The wavefronts propagate through medium m with a speed of c/nm.

There are situations, however, when the wavelength spacing can be forced to be smaller than λo/nm. Such special geometrical situations include the lower refractive index side of an interface at which total internal reflection (TIR) occurs; they also include confinement of the light source to a region smaller than its wavelength, such as very near an excited molecule or the tip of a fine optical fiber. In these cases, light cannot freely propagate and instead becomes exponentially decaying in at least one dimension.

The fundamental physical processes are related among these situations and can be understood most easily by considering the electric field of plane waves and then generalizing to other wavefront shapes. Plane-wave light propagating in a medium of refractive index nm is characterized by a wave vector, km, pointing in the direction of its propagation:

(1.1)

The orientation of the (x, y, z) axes can be arbitrary but is chosen based on the geometry of optical surfaces nearby. The amplitude of km, called the wavenumber, is

(1.2)

where ω is the angular frequency of the particular color of the light, and c is the speed of light in vacuum. Frequency ω is equal everywhere in the optical system, regardless of refractive index.

Because of the constancy of ω and c, the wavenumber, km, and its corresponding wavelength, λm, are real numbers that are fixed for any light in medium m, regardless of direction or proximity to interfaces. For freely propagating light, the spacing between wavefronts along any direction x, y, z of propagation is

(1.3)

The position-dependent part of the plane wave electric field has the form exp(ikm·r. Therefore, the spatial variation of the electric field in, say, the z direction is the sinusoidal function exp(ikmzz).

The relevant feature of Equation (1.2) is that the sum of the squares of the components kmx, kmy, and kmz ? This might happen if the wavefronts in the x–y be negative and kmz thereby be imaginary. The electric field’s spatial dependence in the z direction then becomes an exponentially decaying function exp(−|kmz|z), an evanescent field.

(or, equivalently, squeezed wavelength spacing). These shared mechanisms and individual differences will be examined more closely in the following sections.

Evanescence in Excitation: TIR

In TIR, the wavefront spacing squeeze is a direct consequence of the geometry of refraction at an interface. Plane-wave light approaching a planar interface from a higher index, n3, dielectric toward a lower index, n1 (say, in the x–z plane, with z being the normal to the interface and no component in the y direction so that k1y = k3y = 0; see Figure 1.1), can create an exponentially decaying field (rather than a propagating field) in the lower index medium, provided the incidence angle, θ3, in medium 3 (measured from the normal) is greater than a critical angle θc. From the perspective of ray optics, this critical angle is a straightforward result of Snell’s law, which in general specifies that n1  sin  θ1 = n3  sin  θ3, here evaluated for incidence angle θ3 when the angle of refraction θ1 = 90°, so that

(1.4)

From the perspective of wavefronts as depicted in Figure 1.1, the spacing λ3x of wavefronts along the interface (the x direction) just inside medium 3 is always longer than the natural propagation wavelength λ3 = 2π/k3 in medium 3, because of the non-orthogonal angle with which the x-axis interface cuts the wavefronts. The wavefront spacing λ1x = 2π/k1x in medium 1 on the other side of the interface is always forced to be exactly equal to λ3x because of the requirement to match the periodic boundary conditions imposed by Maxwell’s equations. That common wavefront spacing becomes smaller with increasing θ3 and is given by

(1.5)

As long as λ1x is longer than the propagating light wavelength λ1 = 2π/k1 in medium 1 the periodic boundary condition at the interface can be matched if the light in medium 1 propagates away at some acute refraction angle (given by Snell’s law). But, for a sufficiently large incidence angle θ3 (i.e., greater than θc) the wavelength spacing λ1x along the x direction becomes smaller than the natural propagation wavelength λ1 for medium 1. The corresponding wavenumber k1x for that spacing λ1x is

(1.6)

For θ3 > θc, wavevector component k1x becomes larger than the k1 (= 2π/λ1) permissible for propagating light in medium 1. This situation forces k1z to become imaginary, as seen from Equation (1.2).

FIGURE 1.1   Wavefront spacing in TIR.   (Left panel) Subcritical θ 3 < θ c . (Middle panel) Critical θ 3 = θ c . (Right panel) Supercritical θ 3 > θ c . Wavefronts are shown as heavy solid lines. The plane of incidence is the x – z plane as shown. The wavefront spacing in the x direction, λ 1 x , varies and is progressively squeezed by geometry as θ 3 increases. For θ 3 > θ c , λ 1 x becomes smaller than the spacing λ 1 demanded by freely propagating light in medium 1 (shown as light vertical dashed lines). This squeezing forces the electric field in the z direction to decay exponentially. The heavy dashed arrows indicate propagation direction. A phase shift exists between wavefronts in medium 3 vs. medium 1 for the supercritical case, but it is not shown here in order to clarify the depiction of wavefront spacing.

The TIR evanescent field has four features interesting for experimental techniques: depth, intensity, polarization, and phase.

TIR Theory: Depth

Because the electric field strength z dependence is given by exp(–|kmz|z), the intensity (which is proportional to the square of the electric field strength) in medium 1 has the z dependence exp(–2|kmz|z) = exp(–z/d), where characteristic depth d is (assuming k1y = 0 without loss of generality)

(1.7)

From Equations (1.2), (1.4), and (1.6) we can rewrite this as

(1.8)

For typical refractive indices, depth d ranges from ∼λo for θ3  ≈  θc  +  2°, down to about λo/10 for larger but still easily attainable θ3. This small d is the reason why TIR excitation of fluorescence (TIRF) is useful for selective excitation of surface-proximal molecules in medium 1, cell/substrate contact regions, and membrane-proximal cytoplasmic organelles, while minimizing excitation of background fluorescence originating deeper within the sample.

TIR Theory: Field Strength, Polarization, and Intensity

Complete expressions for each component (in the x, y, z directions as defined above) of the evanescent electric field vector E1 in medium 1 at z = 0 can be derived from boundary conditions imposed by Maxwell’s Equations. Each of these expressions assumes that the incident light electric field E3 is linearly polarized with components E3p and E3s in the p-pol direction (i.e., parallel to the plane of incidence and perpendicular to the propagation direction) and the s-pol direction (i.e., perpendicular to both the planes of incidence and propagation direction), respectively.

(1.9)

where

(1.10)

The angles in the phase factors are

(1.11)

(1.12)

Several qualitative features are implicit in Equations (1.10) to (1.12):

1. The evanescent decay depth (d for intensity and 2d for electric field) is the same for each incident polarization.

2. The amplitude of evanescent electric field E1 and its polarization (i.e., the relative amplitudes of the vectorial components) are strong functions of incidence angle θ3.

3. Given a particular incident E3 and θ3, the components of E1 depend on the ratio of refractive indices n1 and n3 but not on their separate values.

4. An s-pol incident light gives rise to a pure y-polarized evanescent field.

5. A p-pol incident light gives rise to a mix of an x- and z-polarized evanescent field, while its wavefronts propagate along the x direction. This distinguishes a p-pol evanescent field from freely propagating subcritical p) of the p-pol evanescent field is zero at θ3 = θc but increases monotonically in the supercritical range θ3 > θc.

6. The imaginary factor i appears in E1x but not in E1z, meaning that the two components are 90° out of phase and consequently the p-pol evanescent field is elliptically (not linearly) polarized in the plane of incidence.

The evanescent intensities I1p and I1s are proportional to their respective scalar products E∗· E. Given the corresponding incident intensities |E1p|² and |E1s|², the evanescent intensities at z = 0 are

(1.13)

(1.14)

These intensities are proportional to the rate of energy absorption by a fluorophore in the evanescent wave and are plotted vs. θ3 in Figure 1.2 (solid curves). The supercritical evanescent intensity of each polarization monotonically approaches zero as θ3 → 90°. Subcritical angle intensities (corresponding to a propagating refracted ray) are also shown in Figure 1.2; they are completely continuous with their respective supercritical intensities. For each polarization, both the subcritical and supercritical regions reach their (different) maxima at the critical angle. For supercritical angles just above θc, the evanescent s-pol intensity is four times the s-pol incident light intensity |E3s|² (and the p-pol intensity is more than four times |E3p|²). This discrepancy is not a violation of conservation of energy, as the energy in the evanescent field in medium 1 does not escape away from the interface and in fact flows back into medium 3. However, a fluorophore near the interface in medium 1 can tap into that energy and will, in fact, decrement the evanescent intensity slightly by its presence, as would a fluorophore in a propagating light field. In the immediate subcritical range, refracted light does escape the surface region, but the width of the beam (which is always finite) is narrowed by the geometry of refraction, so the increase in its intensity is compensated by its smaller cross-sectional area, and energy is still conserved.

FIGURE 1.2   Evanescent intensity at z = 0 vs. incidence angle θ 3 .   The blue lines are for p -pol, and the red lines are for s -pol. The solid lines are for bare surfaces, and the dashed lines are for surfaces coated with an intermediate layer of 2.4- λ thickness (where λ is the vacuum wavelength of the incident light). The ordinate axis is in multiples of the incident intensity | E 3 p | ² or | E 3 s | ² . Parameters assumed are n 1 = 1.3334 (water), n 2 = 1.42 (intermediate film, if used), and n 3 = 1.5255 (glass coverslip).

The fluorescence emission intensity vs. θ3 profile has been used to deduce the concentration of a fluorophore as a function of distance z from the substrate, because only the lower θ3 angles, with their deeper evanescent field depth, can reach out to fluorophores farther away from the interface.⁶,⁷ But, because the evanescent intensity at z  =  0 is itself a strong function of θ3 that dependence must be taken into account when measuring z-dependent concentration profiles near a TIR surface. (As we shall discuss below, the emission gathering efficiency is also a function of z, a more subtle effect that must also be taken into account.

TIR Resonance in Films

The I vs. θ3 solid line curves of Figure 1.2 (for evanescence at a bare glass/water interface) become much richer if the substrate glass is coated with a thin film of refractive index n2 (see the dashed lines in Figure 1.2).⁸,⁹ If the film is greater than about λo/2 in thickness and its refractive index is intermediate (n3 > n2 > n1), the film can act as a lossy planar waveguide, complete with resonance modes that depend on θ3. If θ3 is such that light propagates into the film from medium 3 but then totally reflects at the interface with medium 1, the totally reflected light will subsequently partially reflect at the n3:n2 interface, return to totally reflect again at the n2:n1 interface (and so on), and thereby set up a pattern of destructive and constructive interference in the film. Because the intensity of the light just inside the film in medium 2 immediately near the n2:n1 interface is proportional to the intensity of the evanescent field just inside medium 1, the resonance-like behavior in the film should be evident in the observed θ3 dependence of fluorescence intensity as excited by the evanescent field in medium 1. From the shape of the fluorescence vs. θ3 curve (perhaps even spatially resolved in a microscope) the thickness and refractive index and possible lateral heterogeneities of the film may be inferred. This phenomenon, as yet unapplied in biophysics, may have applications, for example, in characterizing a multilayer lipid coating supported on a surface. At particular incidence angles θ3 that produce a resonance in the film, the evanescent field in medium 1 can be enhanced in intensity by at least an order of magnitude over what it would be with no film.

Polarized Excitation TIRF

Polarized excitation TIRF is a technique which can uniquely highlight submicroscopic irregularities in the plasma membrane of living cells¹⁰ and in supported lipid bilayers.¹¹,¹² For samples with multiple fluorophores, the effect requires the incorporation of fluorophore into the membrane with a high degree of orientational order. The requirement for ensemble orientational order is eliminated in viewing singly labeled single molecules rather than membranes with highly oriented heavy labeling. Polarized excitation TIR has been successfully used to determine the orientation of single molecules of sparsely labeled F-actin.¹³

Polarized excitation TIRF depends on the fact that the p-pol evanescent field is directed predominantly in the z direction (i.e., normal to the substrate). Note from Equation (1.10) that E1z is typically much larger than E1x for all generally accessible supercritical θ3. If a membrane lying flat upon the substrate is labeled with a fluorophore (such as the membrane probe diI-C18-(3) or diI) whose absorption transition dipole moment orients in the plane of the membrane (the x–y plane), then a p-pol evanescent field will not efficiently excite the fluorophores. But, if the membrane has an indentation (even a submicroscopic one), that indented region will not be parallel to the substrate and thereby can be excited by the p-pol evanescent field. This orientational sensitivity can be separated from local fluorophore concentration changes by dividing the p-pol excited image with a s-pol excited image of the same region.

TIR-FRAP, TIR-FCS, and Two-Photon TIRF

Total internal reflection has been combined with other fluorescence microscopy techniques, and the combinations themselves have important uses and nontrivial theoretical descriptions. TIR excitation can be combined with fluorescence recovery after photobleaching (FRAP) or fluorescence correlation spectroscopy (FCS) to measure: (1) diffusion coefficients of surface-proximal fluorophores, in solution, in model or living cell membranes, in cell cytoplasm, or even within submicroscopic cellular organelles;¹⁴ (2) chemical kinetic rates of binding/unbinding to the surface; and (3) absolute concentrations of fluorophores (in the case of TIR-FCS). The basic theory for these somewhat related techniques¹⁵ and a recent review of TIR-FCS applications and practical experimental protocols¹⁶ have been published. Two-photon TIRF¹⁷ in principle would cut the evanescent characteristic distance d in half. But, because the infrared photons used as an excitation source in this nonlinear technique have about double the wavelength of those typically used in standard single-photon TIRF, that possible advantage is canceled. On the other hand, TIRF sometimes suffers from a low-intensity nonevanescent background due to scattered excitation light, and two-photon TIRF gives a distinct advantage to the brightest (and nonscattering) evanescent portion of the illumination. By further combining two-photon TIRF with TIRF-FRAP, an enhancement of signal can be expected.¹⁸

TIRF Evanescent Field Phase and Interference

The evanescent field in medium 1 is sinusoidally periodic in the x–y plane (the plane of the interface supporting the TIR), with a slight phase shift relative to the incident propagating plane wave in medium 3 that is θ3 dependent. The evanescent field phase has no z dependence, meaning that the wavefronts are perpendicular to the interface and the field can be represented simply as a two-dimensional wave in x–y. Because of the close spacing of wavefronts in the evanescent field, illumination by a pair of intersecting and mutually coherent TIR laser beams can produce a very closely spaced striped interference fringe pattern in the evanescent intensity (provided they have the same evanescent polarization). If the relative azimuthal angle ϕ between the two TIR beams is 180°, then the node-to-node spacing s of the evanescent fringes is given by

(1.15)

Spacing s is not dependent upon the refractive index n1 of the medium (or cell) and can be smaller than the Raleigh resolution limit of the microscope. These fine stripes can be employed in structured illumination,¹⁹ thereby producing an even finer lateral resolution than what is standard for that already super-resolution technique.

Interesting and potentially useful patterns can be produced by interfering more than one pair of TIR beams. Two orthogonal pairs of intersecting coherent TIR beams (i.e., four azimuthal angles spaced at 90°) will produce a checkerboard pattern in the evanescent intensity. In the limit of an infinite number of azimuthal angles (i.e., two-dimensional plane waves of equal strength all converging to a central spot where the phases are the same), we must integrate evanescent two-dimensional plane waves propagating along the surface with wavevectors ksurf from all azimuthal angles ϕ (see Figure 1.3A), each with an amplitude ksurf given by

(1.16)

This is the same wavenumber as k1x in Equation (1.6) except that here the waves are converging from all directions in the surface rather than just propagating along x. The interference among this continuum of waves produces a small bright central spot of sub-wavelength dimension and with increasingly dimmer rings at larger radii, with an intensity pattern given by

(1.17)

where J0 is a zero-order Bessel function, and ρ is a radial vector in the plane of the surface (the x–y plane). The intensity pattern is not an infinitesimal point (i.e., a δ function) because the integration, while done over all the possible two-dimensional azimuthal directions of ksurf given by angle ϕ, is not done over all its possible amplitudes. Rather, the amplitude ksurf is fixed by Equation (1.16).

FIGURE 1.3   TIR from multiple directions.   (A) Schematic view of 12 two-dimensional evanescent plane waves converging to an in-phase center. An infinite number of such two-dimensional plane waves creates a converging circular wave. (B) This circular converging pattern can be produced experimentally by a thin annulus of illumination at the BFP of the microscope objective used for TIRF excitation. The polarization at the BFP must be radial so that the evanescent fields from each plane wave component will be all predominately in the z direction (normal to the TIR surface) so they can mutually interfere. (C) The evanescent intensity theoretically predicted from such annular illumination according to Equation (1.17) . The width of the central maximum to the first minimum is ∼0.26 λ o , assuming the annular ring at the BFP has a radius corresponding to NA   =   1.45, which is easily accessible with a 1.49-NA objective.

Two-dimensional plane waves converging from all azimuthal angles to a central spot can be produced in the lab by illuminating a ring at the objective back focal plane (BFP) of the microscope objective (for objective-based TIRF), which will produce a hollow cone of light of polar angle θ3 with an apex at the sample plane. Constructive interference creating the small TIR spot on the sample plane will occur only if all the component two-dimensional plane waves have the same evanescent polarization. The geometry requires that this evanescent polarization be in the z direction, which will occur only if the illumination ring at the BFP is radially polarized (Figure 1.3B).

The small central spot (Figure 1.3C) has a radius from the central maximum to the first minimum of only 0.26 λo, which is considerably less than the Raleigh resolution limit for a 1.49-NA objective (0.41 λo). Because all of the component plane waves have an incident angle of θ3 the evanescent field depth of the small TIR spot is still given by Equation (1.7). This spot is remarkable because standard coherent single-direction TIR illumination (e.g., a laser beam) cannot produce such a small x–y area of illumination. The tiny size in all three dimensions, achieved by radially polarized ring illumination at the BFP, may be particular useful for defining a small fluorescence correlation spectroscopy (FCS) volume (as FCS works best with very few fluorophores in view) and possibly for scanning stage-based image reconstruction.

Evanescence in Emission: The Fluorophore Near Field

Light emitted from a fluorophore can also show observable effects of evanescence. Even if there are no interfaces nearby, the spacing between wavefronts will be squeezed if the size of the source of light is very small (i.e., sub-wavelength). The result is that a portion of the emitted light field is evanescent in the direction away from the source. This portion is called the near field of the emission, and it has a number of practical consequences and analogies to excitation evanescence.

Near Field of Fluorophore Emission: Theory

surrounding an oscillating dipole vector μ located at the origin in a medium 1 with refractive index ncan be written as follows:

(1.18)

is an outgoing spherical wave oscillating at angular frequency ω, but with amplitude and polarization dependent on the direction and distance to the observation point at r in three-dimensional space. The three terms in the square brackets have increasingly rapid rates of drop-off with distance k1r to the observation point (measured in terms of 2π times the number of wavelengths). The first term, with the factor (k1r)–1, is the standard far-zone light that retains the same integrated energy flow through any sphere centered on the fluorophore and is responsible for all of the propagating emission seen from an isolated fluorophore far (i.e., k1r >> 1) from any interfaces. The next two terms are called the near zone (as opposed to near field, discussed later) because they decay more rapidly with distance.

as an integral over plane waves with some weighting factors.as it interacts with the optical system.

In general, the resolution of a curved wavefront field into component plane waves is called the angular spectrum of plane waves.:

(1.19)

In principle, the integration must be taken over all possible amplitudes and directions of wavevectors k1. But, recall that not all mathematically possible k1 vectors can be used in optics; only those that also satisfy the optics requirement that the sum of the squares of the components k1x,1y,1z can be used. Among those physically possible k, leading to an imaginary k1z and evanescence in the z direction, analogous to the situation in TIRF except with the decay starting at the z position of the fluorophore.

are absolutely necessary to reconstruct the original field. Consider, for example, standard nonevanescent plane waves propagating only in the x–y and thereby k1z  =  0. There is no way that such waves can be added together from all x–y directions simultaneously to produce a sharp singularity at r  =  0 as required by Equation (1.18). Even if the phases of all these propagating waves from all x–y directions are set to add constructively at the origin and are given equal amplitudes, the sum near r = 0 will be a diffuse blur. This is mathematically identical to the case discussed earlier for a coherent ring of TIR illumination, which gives the non-singularity intensity pattern of Equation (1.17) and Figure 1.3C, except that the emission light discussed here propagates away from the center for emission instead of converging toward it. In order to reconstruct the sharp r  =  0 singularity resulting from any of the three terms in Equation (1.18), we require plane waves that are squeezed in their wavefront spacing in x and y and thereby are evanescent in z. This is the origin of the evanescent near field in the immediate vicinity of a fluorophore. The near zone terms of Equation (1.18) are not the sole source of the near field; rather, all three terms, including the far zone term, are responsible for setting up the near field because all three have sharp singularities at r = 0.

How can the near field be detected? If the fluorophore in medium 1 is close to an interface defining the x–y plane with a higher refractive index n3, the tails of these evanescent components will interact with the interface. For x–y wavefront spacings that are not too small, the tails will convert into propagating light in the higher index material and travel away (possibly to a detector) at a large supercritical angle to the interface normal. That supercritical angle corresponds to the incidence angle that hypothetical TIR illumination would trace going in reverse and setting up an evanescent field with the same squeezed wavefront spacing. As the x–y wavefront spacing of an evanescent emission component becomes smaller, the larger becomes the supercritical angle of the propagation in the higher index material. But, for very small x–y spacing in medium 1, no propagating beam with matching spacing at the interface is possible. Such a field will remain evanescent even in the higher index material and will normally not be detected. The higher the refractive index n3, the more evanescent components of the fluorophore near field can be captured, converted into propagating light, and detected.

Near Field of Fluorophores: Collection of Fluorescence and Imaging

Some of the near-field light emitted from a fluorophore converts into a hollow cone of light in a nearby glass substrate, propagating at supercritical angles. That hollow cone can then be captured by a high aperture objective (, is cast into that high-angle hollow cone (or its corresponding supercritical BFP annulus). Far-field light is entirely cast into subcritical angles and a central BFP disk corresponding to NA  <  1.33. The light-collecting advantage of very high-aperture objectives (NA  >  1.33) resides purely in their ability to capture supercritical near-field light. The extra numerical aperture does not help in gathering far-field emission light because none of it propagates in the glass at the supercritical angles.

Figure 1.5 shows a theoretically generated view of the BFP as calculated by the methods of Axelrod⁹ for a dipole lying at z = 0 and oriented parallel to the interface plane. In practice, the BFP can be viewed directly with a minor modification of the imaging optics. Each supercritical polar angle of emission (and radius in the BFP) corresponds to a particular near-field x–y wavefront spacing in the fluorophore near field and, hence, a particular z-direction exponential decay length. But, because the BFP annulus has a finite thickness containing a range of supercritical angles up to the limiting aperture of the objective, the corresponding decay lengths in that annulus also cover a range of decay distances. Therefore, the total light power coming through the BFP supercritical annulus decays as a function of z distance of a fluorophore from