This book offers a complete presentation of the physics, math, and experimental setups for both TIRF and related evanescence microscopies. It covers evanescence in both fluorescence excitation or emission. It also discusses, in detail, the theory, setups, and practical biological/biochemical applications for combinations of evanescence microscopies with other optical techniques such as polarization, photobleaching, correlation spectroscopy, scattering, image enhancement, optical force, two-photon, energy transfer, structured illumination, scanning, quenching, and single molecule detection. Physical and qualitative discussions augment the rigorous math, making the book accessible and interesting to a wide range of audiences with backgrounds from biology to chemistry to physics. The book also contains useful step-by-step guides to building, modifying, and aligning TIRF microscopy systems for specialized purposes.
Key Features:
- Suitable for both basic and advanced levels
- Offers combinations with related techniques
- Relationship of evanescent effects between excitation and emission explained
- Practical guide to the optics as well
Author(s): Daniel Axelrod
Series: Biophysical Society–IOP Series
Publisher: IOP Publishing
Year: 2022
Language: English
Pages: 197
City: Bristol
PRELIMS.pdf
Preface and acknowledgments
Author biography
Daniel Axelrod
CH001.pdf
Chapter 1 Introduction to optical evanescence
1.1 Overview
1.2 Applications to biochemistry and cell biology
1.2.1 Cell/substrate contact regions
1.2.2 Long-term videos of living cells
1.2.3 Secretory granule tracking and exocytosis
1.2.4 Single molecules
1.2.5 Reversibly bound and mobile fluorescent ligands on cells and biosurfaces
1.2.6 Cytoplasmic filaments
1.2.7 Calcium channels and transients
1.2.8 CRISPR
1.2.9 Orientational distributions of fluorescent molecules at a surface
1.2.10 Combinations and comparisons with other microscopy techniques
1.3 Ray picture of total internal reflection
1.4 Maxwell’s equations and wave numbers
1.5 Causes of evanescence: a physical view
1.5.1 Total internal reflection
1.5.2 Small aperture
1.5.3 Waveguides
1.5.4 Near-field emission
Further reading
CH002.pdf
Chapter 2 Total internal reflection theory
2.1 Rays and TIR
2.2 Waves and TIR
2.3 Evanescent intensity
2.4 Finite-width incident beams: the Goos–Hänchen shift
2.5 Reflected intensities
Further reading
CH003.pdf
Chapter 3 Structure in the lower-index material
3.1 Light absorption in medium 1
3.2 Intermediate layers
3.2.1 Field and intensity in medium 1 (z ≥ 0)
3.2.2 Field and intensity in medium 2 (−h < z < 0)
3.2.3 Field and intensity in medium 3 (z < − h)
3.3 Metal films and surface plasmons
3.4 Slab waveguides
3.5 Total internal reflection scattering
3.5.1 Fundamental equations
3.5.2 Parameter definitions
3.5.3 Green’s function solution for the perturbative approach
3.5.4 Inclusion of the local case r = r′
3.5.5 Reporting surface selectivity: intensity and evanescent depth
Further reading
CH004.pdf
Chapter 4 Emission of fluorophores near a surface
4.1 The emission near field: a semi-qualitative view
4.2 Capture of the near field: summary of quantitative theory
4.3 Polarization of the emitted electric field
4.4 Emitted intensity and total power
4.5 Emitted intensity vs polar angle
4.6 Total fluorescence collection through a microscope objective
4.6.1 Single dipole: integration over azimuthal angles
4.6.2 Single dipole: integration over polar angles
4.6.3 Distribution of dipoles
4.7 Pattern at the back focal plane
4.8 Characterization of films with supercritical-emission light
4.9 Effect of metal films on fluorescence emission
4.10 Pattern at the image plane
4.10.1 Approximation of the PSF: the 2D Airy disk
4.10.2 Full calculation of the PSF
4.11 Virtual supercritical angle fluorescence microscopy (vSAF)
4.12 Emission polarization including supercritical light
4.13 SAF/UAF: measurement of the absolute distance between a fluorophore and a surface
4.14 Effect of near-field capture on fluorescence lifetime
Further reading
CH005.pdf
Chapter 5 Optical configurations and setup
5.1 Inverted microscope TIR with prism above
5.2 Inverted microscope TIR with prism below
5.3 Upright microscope TIR with prism below
5.4 Objective-based TIR
5.4.1 Focus at the back focal plane (BFP)
5.4.2 Illumination area in the field of view
5.5 Incidence angle, multicolor, and polarization control
5.5.1 Sample plane, back focal plane, and their equivalents
5.5.2 Polar incidence angle control
5.5.3 Azimuthal incidence angle control
5.5.4 Switching excitation colors
5.5.5 Excitation polarization control
5.6 Alignment
5.7 Rapid chopping between TIR and epi-illumination
5.8 Supercritical-angle fluorescence (SAF) emission setup
5.9 Imaging the back focal plane directly
5.10 Measurement of evanescent field depth
5.11 TIRF–structured illumination microscopy (TIRF–SIM)
5.11.1 Single-spot TIR with converging illumination
5.11.2 Array of TIR spots
5.11.3 Periodic sine-wave pattern
5.11.4 Periodic pattern for image enhancement
5.11.5 Spot TIR with collimated light
Further reading
CH006.pdf
Chapter 6 Applications of TIRF microscopy and its combination with other fluorescence techniques
6.1 Refractive indices in cell cultures
6.2 Axial position and motion of cell components
6.3 Quenching with a metal film
6.4 Image sharpening in TIR
6.5 Polarized excitation TIRF
6.6 Variable-depth TIRF
6.7 Optical force in an evanescent field
6.8 TIR/FCS and TIR/FRAP
6.8.1 Adsorption/desorption chemical kinetics
6.8.2 Characteristic rates
6.8.3 RR
6.8.4 RBND
6.8.5 RSD
6.8.6 RBLD
6.8.7 Limiting solutions for an infinite observation area
6.8.8 Solutions for a finite observation area
6.8.9 TIR/FCS/FRAP to measure the diffusion coefficient in solution
6.8.10 Absolute concentrations: single component
6.8.11 Absolute concentration: mixed components
6.8.12 Higher order TIR-FCS
6.8.13 TIR/FRAP in a sub-resolution confined volume: a spherical secretory granule
6.8.14 Spatially-resolved TIR/FRAP
6.8.15 TIR/FRAP with sine wave
6.9 TIR-continuous photobleaching
6.10 TIR-FRET
6.11 Two-photon TIRF
6.11.1 Two-photon theory
6.11.2 Reduction of scattering effect
6.11.3 Requirement for high intensity
6.11.4 Two-photon sine-wave-pattern TIRF
6.11.5 Two-photon excitation with slab waveguides
Further reading
