Quantitative Bioimaging: An Introduction to Biology, Instrumentation, Experiments, and Data Analysis for Scientists and Engineers (Textbook Series in Physical Sc)

504 Pages·2020·112.237 MB·English

by  Raimund J. Ober, E. Sally Ward, Jerry Chao

Quantitative Bioimaging

Quantitative Bioimaging
An Introduction to Biology, Instrumentation,
Experiments, and Data Analysis
for Scientists and Engineers
Raimund J. Ober
Centre for Cancer Immunology
Faculty of Medicine  
University of Southampton
Southampton, United Kingdom  
E. Sally Ward
Centre for Cancer Immunology
Faculty of Medicine  
University of Southampton
Southampton, United Kingdom
Jerry Chao
Astero Technologies LLC
College Station, Texas, United States
First edition published 2020
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Contents
Preface xvii
Acknowledgments xix
I Introduction 1
Overview 3
1 Then and Now 5
2 Introduction to Two Problems in Cellular Biology 7
2.1 Antibody trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2 Localization experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3 Association experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.4 Dynamic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.5 Iron transport, transferrin, and the transferrin receptor . . . . . . . . . 13
3 Basics of Microscopy Techniques 17
3.1 Optical microscopy for cell biology . . . . . . . . . . . . . . . . . . . . . 17
3.2 Transmitted light microscopy . . . . . . . . . . . . . . . . . . . . . . . . 17
3.3 Fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.3.1 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.2 Layout of an epifluorescence widefield microscope . . . . . . . . 21
3.4 Inverted versus upright microscope . . . . . . . . . . . . . . . . . . . . . 22
3.5 Components of commercial microscopes . . . . . . . . . . . . . . . . . . 23
3.5.1 Light sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.5.2 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.6 Fixed and live cell experiments . . . . . . . . . . . . . . . . . . . . . . . 27
3.7 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.8 A note regarding safety . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4 Introduction to Image Formation and Analysis 29
4.1 Image formation and point spread functions . . . . . . . . . . . . . . . . 29
4.2 Resolution: an elementary introduction . . . . . . . . . . . . . . . . . . . 31
4.3 Modeling and analyzing the data . . . . . . . . . . . . . . . . . . . . . . 34
Notes 37
Exercises 39
II Biology and Chemistry 41
Overview 43
vii
viii Contents
5 From genes to proteins 45
5.1 Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.2 DNA and genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
5.3 How are proteins made? . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.4 Structures of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5.5 Protein structure determination . . . . . . . . . . . . . . . . . . . . . . . 58
6 Antibodies 65
6.1 Structure of antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.2 Variable regions and binding activity . . . . . . . . . . . . . . . . . . . . 67
6.3 Constant regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.4 Antibody production for laboratory and clinical use . . . . . . . . . . . 71
6.4.1 The classical method: hybridoma technology . . . . . . . . . . . 71
6.5 Diagnostic techniques using antibody detection methods . . . . . . . . . 73
6.5.1 Enzyme-linked immunosorbent assay . . . . . . . . . . . . . . . 73
6.5.2 Surfaceplasmonresonanceforthequantitationoftheaffinityofan
interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
7 Cloning of genes for protein expression 77
7.1 Features of expression constructs . . . . . . . . . . . . . . . . . . . . . . 77
7.2 Methods for generating expression plasmids . . . . . . . . . . . . . . . . 78
7.2.1 Restriction enzymes . . . . . . . . . . . . . . . . . . . . . . . . . 78
7.2.2 Polymerase chain reaction. . . . . . . . . . . . . . . . . . . . . . 79
7.2.3 Details of approaches for generating expression plasmids . . . . 79
7.2.4 Transfection of mammalian cells for expression . . . . . . . . . . 86
7.3 Antibody engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
7.3.1 Chimeric antibodies . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.3.2 Humanized antibodies . . . . . . . . . . . . . . . . . . . . . . . . 88
7.3.3 Isolation of V regions . . . . . . . . . . . . . . . . . . . . . . . . 88
8 Principles of Fluorescence 91
8.1 Wave and particle description of light . . . . . . . . . . . . . . . . . . . 91
8.2 Jablonski diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
8.3 Stokes shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
8.4 Photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
8.5 Photophysical characterization of fluorophores . . . . . . . . . . . . . . 94
8.5.1 Quantum yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
8.5.2 Beer-Lambert law, effective absorption cross section and molar ex-
tinction coefficient . . . . . . . . . . . . . . . . . . . . . . . . . . 95
8.5.3 Brightness of a fluorophore . . . . . . . . . . . . . . . . . . . . . 96
8.6 Excitation and emission spectra . . . . . . . . . . . . . . . . . . . . . . . 96
8.7 Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
8.7.1 Chemical fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . 97
8.7.1.1 Labeling of proteins via cysteine or lysine residues . . . 98
8.7.1.2 Labelingofproteinswithfluorophore-conjugatedstrepta-
vidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
8.7.1.3 In situ labeling of proteins in cells using peptide tags . 100
8.7.2 Quantum dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
8.7.2.1 Labeling of proteins with quantum dots. . . . . . . . . 101
8.7.3 Fluorescent proteins . . . . . . . . . . . . . . . . . . . . . . . . . 102
8.7.4 Photoactivatable and photoswitchable fluorescent probes . . . . 105
Contents ix
8.7.5 Other labeling modalities . . . . . . . . . . . . . . . . . . . . . . 105
9 Cells 107
9.1 Cellular structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
9.2 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
9.3 Typical biological systems . . . . . . . . . . . . . . . . . . . . . . . . . . 112
9.3.1 Subcellular trafficking of the Fc receptor, FcRn. . . . . . . . . . 112
9.3.2 Subcellular trafficking of the transferrin receptor . . . . . . . . . 112
9.4 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
9.4.1 Labeling of proteins in fixed cells . . . . . . . . . . . . . . . . . . 113
9.4.2 Sample preparation for typical fixed cell experiments . . . . . . 114
9.4.3 Sample preparation for typical live cell imaging experiments . . 115
Notes 121
Exercises 125
III Optics and Microscopy 127
Overview 129
10 Microscope Designs 131
10.1 Light path for widefield fluorescence microscopy . . . . . . . . . . . . . 131
10.1.1 Infinity-corrected light path. . . . . . . . . . . . . . . . . . . . . 131
10.2 Imaging in three dimensions . . . . . . . . . . . . . . . . . . . . . . . . . 132
10.2.1 Focus control and acquisition of z-stacks . . . . . . . . . . . . . 132
10.2.2 Multifocal plane microscopy . . . . . . . . . . . . . . . . . . . . 133
10.3 Imaging of multiple colors . . . . . . . . . . . . . . . . . . . . . . . . . . 133
10.4 Light path for confocal microscopy . . . . . . . . . . . . . . . . . . . . . 135
10.5 Two-photon excitation microscopy . . . . . . . . . . . . . . . . . . . . . 137
10.6 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
10.6.1 Numerical aperture and immersion medium . . . . . . . . . . . . 140
10.6.2 Corrections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
10.6.3 Transmission efficiency . . . . . . . . . . . . . . . . . . . . . . . 141
10.7 Optical filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
10.7.1 Example: a filter set for a GFP-labeled protein . . . . . . . . . . 145
10.7.2 Imaging of multiple fluorophores . . . . . . . . . . . . . . . . . . 147
10.8 Transmitted light microscopy . . . . . . . . . . . . . . . . . . . . . . . . 151
11 Microscopy Experiments 155
11.1 Fixed cell experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
11.1.1 Localization of FcRn . . . . . . . . . . . . . . . . . . . . . . . . 155
11.1.2 Association experiments with FcRn, EEA1, LAMP1, and transfer-
rin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
11.1.3 Pulse-chase verification of fate of mutated IgG . . . . . . . . . . 158
11.2 Imaging a 3D sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
11.2.1 Acquisition of z-stacks . . . . . . . . . . . . . . . . . . . . . . . . 160
11.2.2 Out-of-focus haze . . . . . . . . . . . . . . . . . . . . . . . . . . 160
11.3 Live cell experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
11.3.1 Example: FcRn-mediated IgG trafficking . . . . . . . . . . . . . 163
11.4 Total internal reflection fluorescence microscopy (TIRFM) . . . . . . . . 164
11.4.1 Objective-based total internal reflection fluorescence microscopy 164