Basic Confocal Microscopy (eBook)

Dr. W. Gray (Jay) Jerome is an Associate Professor of Pathology, Microbiology and Immunology at Vanderbilt University School of Medicine. He has published extensively on microscopy of biological material. His work involves correlating microscopic structural information with biochemical, physiological, and molecular genetic data to provide a comprehensive picture of structure-function relationships. Dr. Jerome is a Fellow of the Microscopy Society of America, a Fellow of the American Association for the Advancement of Science, a Fellow of the American Heart Association and a Past-President of the Microscopy Society of America.  Dr. Robert L. Price is a Research Professor in Cell Biology and Anatomy at the University of South Carolina School of Medicine and Director of the Instrumentation Resource Facility. He has been active in biomedical imaging using confocal microscopy since 1990 and along with Dr. Jerome has taught numerous Confocal Workshops in the United States and Australia.  Dr. Price has received several awards from the University of South Carolina and the Microscopy Society of America, is a Fellow of the Microscopy Society of America, and currently President of the Microscopy Society of America. 

Preface 5
Contents 8
Contributors 10
Chapter 1: Introduction and Historical Perspective 11
1.1 Introduction to the Second Edition of Basic Confocal Microscopy 11
1.2 Why an Introductory Text on Confocal Microscopy? 13
1.3 Historical Perspective 14
1.4 Is the Confocal Hype Legitimate? 19
1.5 The Ten Commandments of Confocal Imaging 20
1.5.1 The Perfect Microscope and the Perfect Microscopist Do Not Exist 21
1.5.2 Confocal Microscopy Is More Than a Confocal Microscope 21
1.5.3 During Specimen Processing the Integrity of the Specimen Must Be Maintained as Much as Possible 23
1.5.4 Photons Are Your Friends and Signal-to-Noise Ratio (SNR) Is King 24
1.5.5 Quantification of Fluorescence in a Confocal Micrograph Is a Challenge and at Best Is Only Semiquantitative 24
1.5.6 Scientific Digital Imaging and Normal Digital Imaging (Family Photography) Are Not the Same 26
1.5.7 Your Image Is Your Data: Garbage in Will Result in Garbage Out 27
1.5.8 The Resolution and Bit Depth Present in a Digital Image Are a One-Way Street 27
1.5.9 The JPEG (Joint Photographic Experts Group) Image File Format Is EVIL but Useful 27
1.5.10 Storage Media Is Essentially Free and Infinite 28
1.6 Summary 29
Literature Cited 29
Chapter 2: The Theory of Fluorescence 31
2.1 Introduction 31
2.2 General Principals 31
2.3 Factors Affecting Fluorescence Emission 37
2.4 Nonlinear Optical Methods 39
2.5 Biological Specificity of Labeling 41
2.6 Recently Developed Fluorescent Probes 43
Literature Cited 46
Chapter 3: Fluorescence Microscopy 47
3.1 Introduction 47
3.2 The Optical Path in a Fluorescence Microscope 48
3.3 Light Sources 49
3.4 Objective Lens Characteristics and Terminology 54
3.4.1 Objective Lens Inscriptions 56
3.4.2 Effects of Chromatic Aberration 60
3.4.3 Refractive Index Mismatch and Spherical Aberrations 64
3.5 Filters 71
3.6 Types of Filters 74
3.6.1 Glass Filters 74
3.6.2 Acousto-optical Tunable Filters (AOTF) 75
3.6.3 Acousto-optical Beam Splitters (AOBS) 76
3.7 Determining an Optimum Filter Combination 76
3.8 Optimizing the Fluorescent Signal 81
Literature Cited 81
Chapter 4: Specimen Preparation 82
4.1 Introduction 82
4.2 Preserved Samples Versus Live Imaging 83
4.3 Working with Fixed Samples 84
4.3.1 Fixation 85
4.4 Working with Very Thick Samples 88
4.4.1 Tissue Sectioning 88
4.4.2 Tissue Clearing 90
4.4.2.1 BABB 91
4.4.2.2 DISCO 92
4.4.2.3 X-CLARITY 93
4.5 Mounting Specimens 95
4.6 Working with Live Cells or Live Tissue 99
4.6.1 Live Cell Imaging Instrument Configuration 100
4.6.2 Live Cell Imaging Modes 101
4.6.3 Selection of Fluorescence Imaging Mode 102
4.6.4 Selection of a Fluorescent Probe 103
4.6.5 Imaging the Living Cells 103
4.6.6 Summary of Live Cell Imaging 104
Literature Cited 105
Chapter 5: Labeling Considerations for Confocal Microscopy 107
5.1 Introduction 107
5.2 Types of Labels 108
5.3 Practical Considerations in Labeling 109
5.4 Fluorescence 110
5.4.1 Photobleaching 110
5.4.2 Autofluorescence 111
5.4.3 Sources of Fluorescence 111
5.5 Desirable Features of Molecules Used as Markers 112
5.5.1 Affinity 112
5.5.2 Avidity 113
5.5.3 Cross-Reactivity 113
5.5.4 Stability 114
5.5.5 Identification 114
5.6 Antibody Generation 115
5.7 Immunoglobulin Classes and Structure 116
5.7.1 Immunoglobulin Subclasses 117
5.7.2 Antibody Structure and Fragments 118
5.7.3 Variable Antibody Domains 120
5.8 Labeling Considerations 123
5.8.1 Review of the Literature 123
5.8.2 Use a Hierarchy of Applications 123
5.8.3 Know the Antibody 124
5.8.4 Antibody Concentration 125
5.8.5 Antibody Conjugation and Level of Fluorescence 125
5.8.6 Use of Second and Third Antibodies 127
5.9 Antibody Sources 128
5.9.1 Polyclonal Whole Molecule 128
5.9.2 Monoclonal Whole Antibody 129
5.9.3 Antibody Fragments 130
5.9.4 Second Antibody 130
5.9.5 Control Antibody 131
5.10 Conditions of Labeling 131
5.10.1 Maintain Antigenicity 131
5.10.2 Maintain Optimal Affinity and Avidity 132
5.10.3 Minimize Nonspecific Binding 132
5.10.4 Preserve the Specimen Morphology 132
5.11 Considerations in Labeling with Ligands 133
5.12 Live Cell Labeling 135
5.13 Labeling with Particles 135
5.14 Sequential Versus Simultaneous Labeling 136
5.15 Troubleshooting 137
5.15.1 “Operator Error” 137
5.15.2 Background Labeling 138
5.15.3 Specificity and Affinity 139
5.16 Masked Epitopes and Antigen Retrieval 140
5.17 Fixed Epitopes 141
Literature Cited 141
Chapter 6: Digital Imaging 143
6.1 Introduction 143
6.2 Analog Versus Digital Information 143
6.3 Pixels and Pixel Size (Spatial Resolution) 145
6.4 Pixel Density 149
6.5 Pixel Histogram Analysis 151
6.5.1 Histogram Stretch 151
6.5.1.1 Digital Image Gamma 152
6.5.2 Avoid Digital Contrast and Brightness Functions 154
6.6 Use of Histogram Stretch and Gamma Changes in Confocal Imaging 155
6.7 Image Voxels 156
6.8 Color Images 157
6.9 File Formats 159
Literature Cited 161
Chapter 7: Confocal Digital Image Capture 162
7.1 Introduction 162
7.2 Basics of Optical Microscopy Resolution 163
7.2.1 Lateral Spatial Resolution 164
7.2.2 Lateral Contrast Resolution 167
7.2.3 Lateral Contrast Transfer Function 170
7.3 Image Production and Resolution in a Scanning Confocal Microscope 171
7.3.1 Image Production in a Single Point Scanning Confocal Microscope 173
7.3.2 Image Production in a Multipoint Scanning Confocal Microscope 176
7.3.3 Matching Microscope Image Lateral Resolution to Photodiode Size for cCCD and sCMOS 182
7.3.4 Effect of Missampling 184
7.4 CDD Dynamic Range and Array Size 185
7.5 Image Capture in 3-D 187
7.6 A Word About Color Image Capture: Don’t! 191
7.7 Conclusions 193
Literature Cited 193
Chapter 8: Types of Confocal Instruments: Basic Principles and Advantages and Disadvantages 194
8.1 Introduction 194
8.2 Single-Photon Point-Scanning Confocal Microscopes 195
8.2.1 Limitations of Single-Photon Confocal Systems: Cost 198
8.2.2 Limitations of Single-Photon Confocal Systems: Difficult to Operate 198
8.2.3 Limitations of Single-Photon Confocal Systems: Speed of Acquisition 198
8.2.4 Limitations of Single-Photon Confocal Systems: Photobleaching and Phototoxicity 199
8.2.5 Limitations of Single-Photon Confocal Systems: Resolution 200
8.3 Multiphoton Point-Scanning Confocal Systems 204
8.4 Spinning Disk Systems 207
8.4.1 The Nipkow Disk 207
8.4.2 Nipkow Disk Pinhole Size 208
8.4.3 Nipkow Disk Pinhole Spacing 211
8.4.4 The Petran Microscope 212
8.4.5 The Xiao and Kino Microscope 213
8.4.6 The Corle Microscope 215
8.4.7 The Yokogawa Spinning Disk Confocal Microscope 217
8.4.8 Slit Scanning Systems 218
8.4.9 Image Collection in Spinning Disk Systems 219
Literature Cited 219
Chapter 9: Setting the Confocal Microscope Operating Parameters 221
9.1 Introduction 221
9.2 The Leica SP8 STP8000 Controller and USB Control Panel 226
9.3 Test Specimens and System Performance 227
9.4 Definition of a Good Confocal Image 230
9.5 The Opening Screen 231
9.6 The Configuration Screen 233
9.6.1 Stage Configuration 233
9.6.2 Instrument Parameter Settings (IPS) 233
9.6.3 Beam Path 234
9.6.4 Laser Configuration 236
9.6.5 USB Control Panel 237
9.6.6 Objective Configuration 238
9.6.7 Hardware Settings 239
9.6.8 User Configuration and Memory Management 240
9.6.9 Dye Database 240
9.7 The Acquire Menu 242
9.7.1 Acquire Window Left Panel: Open Projects 244
9.7.2 Acquire Window Acquisition Mode: Resolution 244
9.7.3 Acquire Window Acquisition Mode: Scan Speed 248
9.7.4 Acquire Window Acquisition Mode: Bidirectional Scanning 250
9.7.5 Acquire Window Acquisition Mode: Zoom and Rotation 251
9.7.6 Acquire Window Acquisition Mode: Optical Section Thickness and Pinhole Setting 253
9.7.7 Acquire Window Acquisition Mode: Line and Frame Average and Accumulation 257
9.7.8 Acquire Window Acquisition Mode: Auto Gain 260
9.7.9 Acquire Window Acquisition Mode: Z- Series Collection 260
9.7.10 Acquire Window Acquisition Mode: Scan Window Simultaneous and Sequential Modes 263
9.8 Acquire Menu: Center Window 264
9.9 Creating Protocols 269
9.9.1 A Simultaneous Protocol for Alexa 488 and Cy5 Fluorochromes 271
9.9.2 Sequential Protocol for Alexa 488, Cy3, and Cy5 272
9.10 Image Display Window 276
9.11 Summary 281
Literature Cited 283
Chapter 10: 3D Reconstruction of Confocal Image Data 284
10.1 Introduction 284
10.2 Why Reconstruct? 284
10.3 Defining the 3D Space Using Images 286
10.3.1 Perspective 286
10.3.2 Voxels 287
10.3.3 Resolution 289
10.4 Types of 3D Reconstruction 291
10.4.1 Maximum Projections 291
10.4.2 Volume Rendering 294
10.4.3 Surface Reconstruction 296
10.5 The Steps to Reconstruction 299
10.5.1 Planning 299
10.5.2 Acquisition 301
10.5.2.1 Deconvolution 301
10.5.3 Alignment 301
10.5.4 Adjustment 302
10.5.4.1 Contrast-Limited Adaptive Histogram Equalization (CLAHE) 305
10.5.4.2 Z-Drop 305
10.5.5 Segmentation 306
10.5.6 Modeling and Visualization 308
10.5.7 Measurement 309
Literature Cited 312
Chapter 11: Analysis of Image Similarity and Relationship 313
11.1 Introduction 313
11.2 Colocalization: The Analysis of Similarity in Two Grayscale Images 314
11.3 Resolution 314
11.4 Color Perception and Colorimetric Display 316
11.5 Optimization of Image Quality 318
11.6 Object-Based Overlap Analysis 319
11.7 Scatterplot Analysis 322
11.8 Image Similarity Coefficients 323
11.8.1 Pearson’s Correlation Coefficient 323
11.8.2 Manders’ Overlap Coefficients (MOCs) 325
11.8.3 Setting Appropriate and Unbiased Intensity Threshold Level 330
11.8.4 Expanding Correlation Analysis with Spearman’s Rank Correlation Coefficient 331
11.9 Global Factors Affecting Molecular Clustering 333
11.10 Take-Home Message 334
References 335
Chapter 12: Ethics and Resources 338
12.1 Introduction 338
12.2 Imaging Ethics 339
12.3 Journal and Office of Research Integrity Guidelines 340
12.4 Microscopy Society of America Statement on Ethical Digital Imaging 341
12.5 Available Resources 342
Additional Literature Cited 344
Glossary (Terms Are Defined with Respect to Confocal Imaging) 346
Index 358