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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
by CRC Press
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and by CRC Press

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© 2021 Taylor & Francis Group, LLC

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To our families.
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 Surface plasmon resonance for the quantitation of the affinity of an
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 Labeling of proteins with fluorophore-conjugated strepta-
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
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