Optical Evanescence Microscopy (TIRF): Total Internal Reflection Excitation and Near Field Emission

198 Pages·2022·28.167 MB·English

by  Daniel Axelrod

Total Internal Reflection
Fluorescence (TIRF) and
Evanescence Microscopies
Biophysical Society–IOP Series
Committee Chairperson
Les Satin
University of Michigan, USA
Editorial Advisory Board Members
Geoffrey Winston Abbott Da-Neng Wang
UC Irvine, USA New York University, USA
Mibel Aguilar Kathleen Hall
Monash University, Australia WashingtonUniversityinStLouis,USA
Cynthia Czajkowski David Sept
University of Wisconsin, USA University of Michigan, USA
Miriam Goodman Andrea Meredith
Stanford University, USA University of Maryland, USA
Jim Sellers Leslie M Loew
NIH, USA University of Connecticut School of
Medicine, USA
Joe Howard
Yale University, USA
Meyer Jackson
University of Wisconsin, USA
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Total Internal Reflection
Fluorescence (TIRF) and
Evanescence Microscopies
Daniel Axelrod
Department of Physics, LSA Biophysics, University of Michigan,
Ann Arbor, MI, USA
IOP Publishing, Bristol, UK
ªIOPPublishingLtd2022
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sections77and78oftheCopyright,DesignsandPatentsAct1988.
ISBN 978-0-7503-3351-1(ebook)
ISBN 978-0-7503-3349-8(print)
ISBN 978-0-7503-3352-8(myPrint)
ISBN 978-0-7503-3350-4(mobi)
DOI 10.1088/978-0-7503-3351-1
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Contents
Preface and acknowledgments ix
Author biography xi
1 Introduction to optical evanescence 1-1
1.1 Overview 1-1
1.2 Applications to biochemistry and cell biology 1-2
1.2.1 Cell/substrate contact regions 1-2
1.2.2 Long-term videos of living cells 1-2
1.2.3 Secretory granule tracking and exocytosis 1-2
1.2.4 Single molecules 1-3
1.2.5 Reversibly bound and mobile fluorescent ligands on cells and 1-4
biosurfaces
1.2.6 Cytoplasmic filaments 1-4
1.2.7 Calcium channels and transients 1-5
1.2.8 CRISPR 1-5
1.2.9 Orientational distributions of fluorescent molecules at a surface 1-5
1.2.10 Combinations and comparisons with other microscopy 1-5
techniques
1.3 Ray picture of total internal reflection 1-7
1.4 Maxwell’s equations and wave numbers 1-7
1.5 Causes of evanescence: a physical view 1-9
1.5.1 Total internal reflection 1-10
1.5.2 Small aperture 1-11
1.5.3 Waveguides 1-13
1.5.4 Near-field emission 1-13
Further reading 1-14
2 Total internal reflection theory 2-1
2.1 Rays and TIR 2-1
2.2 Waves and TIR 2-2
2.3 Evanescent intensity 2-6
2.4 Finite-width incident beams: the Goos–Hänchen shift 2-9
2.5 Reflected intensities 2-11
Further reading 2-14
v
TotalInternalReflectionFluorescence(TIRF)andEvanescenceMicroscopies
3 Structure in the lower-index material 3-1
3.1 Light absorption in medium 1 3-1
3.2 Intermediate layers 3-3
3.2.1 Field and intensity in medium 1 (z ≥ 0) 3-5
3.2.2 Field and intensity in medium 2 (−h < z < 0) 3-9
3.2.3 Field and intensity in medium 3 (z < − h) 3-10
3.3 Metal films and surface plasmons 3-11
3.4 Slab waveguides 3-13
3.5 Total internal reflection scattering 3-17
3.5.1 Fundamental equations 3-18
3.5.2 Parameter definitions 3-20
3.5.3 Green’s function solution for the perturbative approach 3-21
3.5.4 Inclusion of the local case r = r′ 3.22
3.5.5 Reporting surface selectivity: intensity and 3-24
evanescent depth
Further reading 3-26
4 Emission of fluorophores near a surface 4-1
4.1 The emission near field: a semi-qualitative view 4-2
4.2 Capture of the near field: summary of quantitative theory 4-5
4.3 Polarization of the emitted electric field 4-9
4.4 Emitted intensity and total power 4-10
4.5 Emitted intensity vs polar angle 4-12
4.6 Total fluorescence collection through a microscope objective 4-15
4.6.1 Single dipole: integration over azimuthal angles 4-15
4.6.2 Single dipole: integration over polar angles 4-16
4.6.3 Distribution of dipoles 4-17
4.7 Pattern at the back focal plane 4-19
4.8 Characterization of films with supercritical-emission light 4-22
4.9 Effect of metal films on fluorescence emission 4-22
4.10 Pattern at the image plane 4-26
4.10.1 Approximation of the PSF: the 2D Airy disk 4-26
4.10.2 Full calculation of the PSF 4-32
4.11 Virtual supercritical angle fluorescence microscopy (vSAF) 4-33
4.12 Emission polarization including supercritical light 4-34
4.13 SAF/UAF: measurement of the absolute distance between 4-35
a fluorophore and a surface
vi
TotalInternalReflectionFluorescence(TIRF)andEvanescenceMicroscopies
4.14 Effect of near-field capture on fluorescence lifetime 4-36
Further reading 4-37
5 Optical configurations and setup 5-1
5.1 Inverted microscope TIR with prism above 5-2
5.2 Inverted microscope TIR with prism below 5-5
5.3 Upright microscope TIR with prism below 5-6
5.4 Objective-based TIR 5-7
5.4.1 Focus at the back focal plane (BFP) 5-7
5.4.2 Illumination area in the field of view 5-10
5.5 Incidence angle, multicolor, and polarization control 5-11
5.5.1 Sample plane, back focal plane, and their equivalents 5-12
5.5.2 Polar incidence angle control 5-12
5.5.3 Azimuthal incidence angle control 5-13
5.5.4 Switching excitation colors 5-14
5.5.5 Excitation polarization control 5-15
5.6 Alignment 5-15
5.7 Rapid chopping between TIR and epi-illumination 5-17
5.8 Supercritical-angle fluorescence (SAF) emission setup 5-17
5.9 Imaging the back focal plane directly 5-19
5.10 Measurement of evanescent field depth 5-20
5.11 TIRF–structured illumination microscopy (TIRF–SIM) 5-22
5.11.1 Single-spot TIR with converging illumination 5-22
5.11.2 Array of TIR spots 5-25
5.11.3 Periodic sine-wave pattern 5-26
5.11.4 Periodic pattern for image enhancement 5-27
5.11.5 Spot TIR with collimated light 5-29
Further reading 5-31
6 Applications of TIRF microscopy and its combination 6-1
with other fluorescence techniques
6.1 Refractive indices in cell cultures 6-1
6.2 Axial position and motion of cell components 6-3
6.3 Quenching with a metal film 6-10
6.4 Image sharpening in TIR 6-11
6.5 Polarized excitation TIRF 6-12
6.6 Variable-depth TIRF 6-17
vii
TotalInternalReflectionFluorescence(TIRF)andEvanescenceMicroscopies
6.7 Optical force in an evanescent field 6-18
6.8 TIR/FCS and TIR/FRAP 6-20
6.8.1 Adsorption/desorption chemical kinetics 6-20
6.8.2 Characteristic rates 6-26
6.8.3 R 6-28
R
6.8.4 R 6-28
BND
6.8.5 R 6-29
SD
6.8.6 R 6-29
BLD
6.8.7 Limiting solutions for an infinite observation area 6-29
6.8.8 Solutions for a finite observation area 6-30
6.8.9 TIR/FCS/FRAP to measure the diffusion coefficient in solution 6-32
6.8.10 Absolute concentrations: single component 6-33
6.8.11 Absolute concentration: mixed components 6-35
6.8.12 Higher order TIR-FCS 6-37
6.8.13 TIR/FRAP in a sub-resolution confined volume: a spherical 6-37
secretory granule
6.8.14 Spatially-resolved TIR/FRAP 6-45
6.8.15 TIR/FRAP with sine wave 6-46
6.9 TIR-continuous photobleaching 6-48
6.10 TIR-FRET 6-51
6.11 Two-photon TIRF 6-51
6.11.1 Two-photon theory 6-52
6.11.2 Reduction of scattering effect 6-57
6.11.3 Requirement for high intensity 6-58
6.11.4 Two-photon sine-wave-pattern TIRF 6-59
6.11.5 Two-photon excitation with slab waveguides 6-60
Further reading 6-61
viii
Preface and acknowledgments
This book is intended as a ‘one-stop shopping’ exposition of the theory and setup
details of evanescence microscopy—in particular, total internal reflection fluores-
cence (TIRF) and supercritical-angle fluorescence. It also includes discussions of
manyofthecombinationsofTIRFwithothertechniques.Everysection‘startsfrom
the beginning’ so it should be readable by anyone with a science background that
includes some physics and math. Most sections proceed to fairly advanced details.
Hopefully,thiswillmakethebookusableasaresearchreferenceaswellasatrigger
for new ideas. In addition to the formal math, a physically intuitive explanation is
provided wherever possible. Also, the topics and explanations here will hopefully
not go out of date too soon, because they focus on the fundamentals underpinning
the techniques. If any important techniques of optical evanescent microscopy have
notbeenadequatelycovered,Iapologize,andIwouldverymuchliketohearabout
them and possibly include them in future updates if necessary.
ThereisonethingthatthisTIRFbookisnot.Itisnotintendedasareviewofthe
many cell biology questions to which TIRF has been applied. The biological
applications discussed here are included only because they illustrate the technique
employedinstudyingthem.Therefore, thereferencelists(‘FurtherReading’ listsat
theendofeachchapter)arelimitedtoasubsetofpublishedworksthatprovidemore
detail on the theory and techniques.
Many people have contributed to the writing in this book, whether intentionally
or inadvertently. First, of course, are those people who are direct coauthors of
particular sections, without whom those sections would not even exist here. This
group (listed here with their most recent affiliations) includes my former graduate
students in Physics at the University of Michigan, Nancy L Thompson (Dept. of
Chemistry,UniversityofNorthCarolina,ChapelHill)andEdwardHHellen(Dept.
of Physics, University of North Carolina, Greensboro). It also includes Geneva M
Omann (Dept. of Biological Chemistry, University of Michigan) and Jeremy J
Axelrod (Dept. of Physics, University of California, Berkeley). They were inten-
tional contributors. An important but inadvertent contributor was Melvyn N
Kronick, who I only fortuitously knew during a one week overlap while he was
starting a postdoc at Berkeley just as I was finishing up there in Melvin P Klein’s
researchgroup.MelvynintroducedmetotheexistenceofTIRF(non-microscopic),
the topic of his PhD thesis at Stanford in William Little’s research group.
IalsothankDrJeromeMertz(BostonUniversity),DrPaulSelvin(Universityof
Illinois), Dr Bethe Scalettar (Lewis and Clark College), and Dr James Abney
(Portland, OR) for useful discussions about diverse aspects of fluorescence micro-
scopy, and Sharon Axelrod for help with a literature search of TIRF applications.
I have benefited from collaborations with many researchers at the University of
Michigan,someofwhomhavebeeninvolvedinTIRFapplicationswithmygroup,
andwhogenerallyprovidedthebiologicalexpertisethatIlack.Mylongtimefriend
andcolleagueRonaldVHolz(Dept.ofPharmacology)wasabsolutelyinvaluablein
enrichingmyscientificcareer(andextendingitwellpast‘retirement’).Ourpostdocs
ix