E-Book, Englisch, 371 Seiten, eBook
Jerome / Price Basic Confocal Microscopy
2. Auflage 2018
ISBN: 978-3-319-97454-5
Verlag: Springer International Publishing
Format: PDF
Kopierschutz: 1 - PDF Watermark
E-Book, Englisch, 371 Seiten, eBook
ISBN: 978-3-319-97454-5
Verlag: Springer International Publishing
Format: PDF
Kopierschutz: 1 - PDF Watermark
Basic Confocal Microscopy, Second Edition builds on the successful first edition by keeping the same format and reflecting relevant changes and recent developments in this still-burgeoning field. This format is based on the Confocal Microscopy Workshop that has been taught by several of the authors for nearly 20 years and remains a popular workshop for gaining basic skills in confocal microscopy. While much of the information concerning fluorescence and confocal microscopy that made the first edition a success has not changed in the six years since the book was first published, confocal imaging is an evolving field and recent advances in detector technology, operating software, tissue preparation and clearing, image analysis, and more have been updated to reflect this. Several of these advances are now considered routine in many laboratories, and others such as super resolution techniques built on confocal technology are becoming widely available.
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.
Zielgruppe
Research
Autoren/Hrsg.
Weitere Infos & Material
1;Preface;5
2;Contents;8
3;Contributors;10
4;Chapter 1: Introduction and Historical Perspective;11
4.1;1.1 Introduction to the Second Edition of Basic Confocal Microscopy;11
4.2;1.2 Why an Introductory Text on Confocal Microscopy?;13
4.3;1.3 Historical Perspective;14
4.4;1.4 Is the Confocal Hype Legitimate?;19
4.5;1.5 The Ten Commandments of Confocal Imaging;20
4.5.1;1.5.1 The Perfect Microscope and the Perfect Microscopist Do Not Exist;21
4.5.2;1.5.2 Confocal Microscopy Is More Than a Confocal Microscope;21
4.5.3;1.5.3 During Specimen Processing the Integrity of the Specimen Must Be Maintained as Much as Possible;23
4.5.4;1.5.4 Photons Are Your Friends and Signal-to-Noise Ratio (SNR) Is King;24
4.5.5;1.5.5 Quantification of Fluorescence in a Confocal Micrograph Is a Challenge and at Best Is Only Semiquantitative;24
4.5.6;1.5.6 Scientific Digital Imaging and Normal Digital Imaging (Family Photography) Are Not the Same;26
4.5.7;1.5.7 Your Image Is Your Data: Garbage in Will Result in Garbage Out;27
4.5.8;1.5.8 The Resolution and Bit Depth Present in a Digital Image Are a One-Way Street;27
4.5.9;1.5.9 The JPEG (Joint Photographic Experts Group) Image File Format Is EVIL but Useful;27
4.5.10;1.5.10 Storage Media Is Essentially Free and Infinite;28
4.6;1.6 Summary;29
4.7;Literature Cited;29
5;Chapter 2: The Theory of Fluorescence;31
5.1;2.1 Introduction;31
5.2;2.2 General Principals;31
5.3;2.3 Factors Affecting Fluorescence Emission;37
5.4;2.4 Nonlinear Optical Methods;39
5.5;2.5 Biological Specificity of Labeling;41
5.6;2.6 Recently Developed Fluorescent Probes;43
5.7;Literature Cited;46
6;Chapter 3: Fluorescence Microscopy;47
6.1;3.1 Introduction;47
6.2;3.2 The Optical Path in a Fluorescence Microscope;48
6.3;3.3 Light Sources;49
6.4;3.4 Objective Lens Characteristics and Terminology;54
6.4.1;3.4.1 Objective Lens Inscriptions;56
6.4.2;3.4.2 Effects of Chromatic Aberration;60
6.4.3;3.4.3 Refractive Index Mismatch and Spherical Aberrations;64
6.5;3.5 Filters;71
6.6;3.6 Types of Filters;74
6.6.1;3.6.1 Glass Filters;74
6.6.2;3.6.2 Acousto-optical Tunable Filters (AOTF);75
6.6.3;3.6.3 Acousto-optical Beam Splitters (AOBS);76
6.7;3.7 Determining an Optimum Filter Combination;76
6.8;3.8 Optimizing the Fluorescent Signal;81
6.9;Literature Cited;81
7;Chapter 4: Specimen Preparation;82
7.1;4.1 Introduction;82
7.2;4.2 Preserved Samples Versus Live Imaging;83
7.3;4.3 Working with Fixed Samples;84
7.3.1;4.3.1 Fixation;85
7.4;4.4 Working with Very Thick Samples;88
7.4.1;4.4.1 Tissue Sectioning;88
7.4.2;4.4.2 Tissue Clearing;90
7.4.2.1;4.4.2.1 BABB;91
7.4.2.2;4.4.2.2 DISCO;92
7.4.2.3;4.4.2.3 X-CLARITY;93
7.5;4.5 Mounting Specimens;95
7.6;4.6 Working with Live Cells or Live Tissue;99
7.6.1;4.6.1 Live Cell Imaging Instrument Configuration;100
7.6.2;4.6.2 Live Cell Imaging Modes;101
7.6.3;4.6.3 Selection of Fluorescence Imaging Mode;102
7.6.4;4.6.4 Selection of a Fluorescent Probe;103
7.6.5;4.6.5 Imaging the Living Cells;103
7.6.6;4.6.6 Summary of Live Cell Imaging;104
7.7;Literature Cited;105
8;Chapter 5: Labeling Considerations for Confocal Microscopy;107
8.1;5.1 Introduction;107
8.2;5.2 Types of Labels;108
8.3;5.3 Practical Considerations in Labeling;109
8.4;5.4 Fluorescence;110
8.4.1;5.4.1 Photobleaching;110
8.4.2;5.4.2 Autofluorescence;111
8.4.3;5.4.3 Sources of Fluorescence;111
8.5;5.5 Desirable Features of Molecules Used as Markers;112
8.5.1;5.5.1 Affinity;112
8.5.2;5.5.2 Avidity;113
8.5.3;5.5.3 Cross-Reactivity;113
8.5.4;5.5.4 Stability;114
8.5.5;5.5.5 Identification;114
8.6;5.6 Antibody Generation;115
8.7;5.7 Immunoglobulin Classes and Structure;116
8.7.1;5.7.1 Immunoglobulin Subclasses;117
8.7.2;5.7.2 Antibody Structure and Fragments;118
8.7.3;5.7.3 Variable Antibody Domains;120
8.8;5.8 Labeling Considerations;123
8.8.1;5.8.1 Review of the Literature;123
8.8.2;5.8.2 Use a Hierarchy of Applications;123
8.8.3;5.8.3 Know the Antibody;124
8.8.4;5.8.4 Antibody Concentration;125
8.8.5;5.8.5 Antibody Conjugation and Level of Fluorescence;125
8.8.6;5.8.6 Use of Second and Third Antibodies;127
8.9;5.9 Antibody Sources;128
8.9.1;5.9.1 Polyclonal Whole Molecule;128
8.9.2;5.9.2 Monoclonal Whole Antibody;129
8.9.3;5.9.3 Antibody Fragments;130
8.9.4;5.9.4 Second Antibody;130
8.9.5;5.9.5 Control Antibody;131
8.10;5.10 Conditions of Labeling;131
8.10.1;5.10.1 Maintain Antigenicity;131
8.10.2;5.10.2 Maintain Optimal Affinity and Avidity;132
8.10.3;5.10.3 Minimize Nonspecific Binding;132
8.10.4;5.10.4 Preserve the Specimen Morphology;132
8.11;5.11 Considerations in Labeling with Ligands;133
8.12;5.12 Live Cell Labeling;135
8.13;5.13 Labeling with Particles;135
8.14;5.14 Sequential Versus Simultaneous Labeling;136
8.15;5.15 Troubleshooting;137
8.15.1;5.15.1 “Operator Error”;137
8.15.2;5.15.2 Background Labeling;138
8.15.3;5.15.3 Specificity and Affinity;139
8.16;5.16 Masked Epitopes and Antigen Retrieval;140
8.17;5.17 Fixed Epitopes;141
8.18;Literature Cited;141
9;Chapter 6: Digital Imaging;143
9.1;6.1 Introduction;143
9.2;6.2 Analog Versus Digital Information;143
9.3;6.3 Pixels and Pixel Size (Spatial Resolution);145
9.4;6.4 Pixel Density;149
9.5;6.5 Pixel Histogram Analysis;151
9.5.1;6.5.1 Histogram Stretch;151
9.5.1.1;6.5.1.1 Digital Image Gamma;152
9.5.2;6.5.2 Avoid Digital Contrast and Brightness Functions;154
9.6;6.6 Use of Histogram Stretch and Gamma Changes in Confocal Imaging;155
9.7;6.7 Image Voxels;156
9.8;6.8 Color Images;157
9.9;6.9 File Formats;159
9.10;Literature Cited;161
10;Chapter 7: Confocal Digital Image Capture;162
10.1;7.1 Introduction;162
10.2;7.2 Basics of Optical Microscopy Resolution;163
10.2.1;7.2.1 Lateral Spatial Resolution;164
10.2.2;7.2.2 Lateral Contrast Resolution;167
10.2.3;7.2.3 Lateral Contrast Transfer Function;170
10.3;7.3 Image Production and Resolution in a Scanning Confocal Microscope;171
10.3.1;7.3.1 Image Production in a Single Point Scanning Confocal Microscope;173
10.3.2;7.3.2 Image Production in a Multipoint Scanning Confocal Microscope;176
10.3.3;7.3.3 Matching Microscope Image Lateral Resolution to Photodiode Size for cCCD and sCMOS;182
10.3.4;7.3.4 Effect of Missampling;184
10.4;7.4 CDD Dynamic Range and Array Size;185
10.5;7.5 Image Capture in 3-D;187
10.6;7.6 A Word About Color Image Capture: Don’t!;191
10.7;7.7 Conclusions;193
10.8;Literature Cited;193
11;Chapter 8: Types of Confocal Instruments: Basic Principles and Advantages and Disadvantages;194
11.1;8.1 Introduction;194
11.2;8.2 Single-Photon Point-Scanning Confocal Microscopes;195
11.2.1;8.2.1 Limitations of Single-Photon Confocal Systems: Cost;198
11.2.2;8.2.2 Limitations of Single-Photon Confocal Systems: Difficult to Operate;198
11.2.3;8.2.3 Limitations of Single-Photon Confocal Systems: Speed of Acquisition;198
11.2.4;8.2.4 Limitations of Single-Photon Confocal Systems: Photobleaching and Phototoxicity;199
11.2.5;8.2.5 Limitations of Single-Photon Confocal Systems: Resolution;200
11.3;8.3 Multiphoton Point-Scanning Confocal Systems;204
11.4;8.4 Spinning Disk Systems;207
11.4.1;8.4.1 The Nipkow Disk;207
11.4.2;8.4.2 Nipkow Disk Pinhole Size;208
11.4.3;8.4.3 Nipkow Disk Pinhole Spacing;211
11.4.4;8.4.4 The Petran Microscope;212
11.4.5;8.4.5 The Xiao and Kino Microscope;213
11.4.6;8.4.6 The Corle Microscope;215
11.4.7;8.4.7 The Yokogawa Spinning Disk Confocal Microscope;217
11.4.8;8.4.8 Slit Scanning Systems;218
11.4.9;8.4.9 Image Collection in Spinning Disk Systems;219
11.5;Literature Cited;219
12;Chapter 9: Setting the Confocal Microscope Operating Parameters;221
12.1;9.1 Introduction;221
12.2;9.2 The Leica SP8 STP8000 Controller and USB Control Panel;226
12.3;9.3 Test Specimens and System Performance;227
12.4;9.4 Definition of a Good Confocal Image;230
12.5;9.5 The Opening Screen;231
12.6;9.6 The Configuration Screen;233
12.6.1;9.6.1 Stage Configuration;233
12.6.2;9.6.2 Instrument Parameter Settings (IPS);233
12.6.3;9.6.3 Beam Path;234
12.6.4;9.6.4 Laser Configuration;236
12.6.5;9.6.5 USB Control Panel;237
12.6.6;9.6.6 Objective Configuration;238
12.6.7;9.6.7 Hardware Settings;239
12.6.8;9.6.8 User Configuration and Memory Management;240
12.6.9;9.6.9 Dye Database;240
12.7;9.7 The Acquire Menu;242
12.7.1;9.7.1 Acquire Window Left Panel: Open Projects;244
12.7.2;9.7.2 Acquire Window Acquisition Mode: Resolution;244
12.7.3;9.7.3 Acquire Window Acquisition Mode: Scan Speed;248
12.7.4;9.7.4 Acquire Window Acquisition Mode: Bidirectional Scanning;250
12.7.5;9.7.5 Acquire Window Acquisition Mode: Zoom and Rotation;251
12.7.6;9.7.6 Acquire Window Acquisition Mode: Optical Section Thickness and Pinhole Setting;253
12.7.7;9.7.7 Acquire Window Acquisition Mode: Line and Frame Average and Accumulation;257
12.7.8;9.7.8 Acquire Window Acquisition Mode: Auto Gain;260
12.7.9;9.7.9 Acquire Window Acquisition Mode: Z- Series Collection;260
12.7.10;9.7.10 Acquire Window Acquisition Mode: Scan Window Simultaneous and Sequential Modes;263
12.8;9.8 Acquire Menu: Center Window;264
12.9;9.9 Creating Protocols;269
12.9.1;9.9.1 A Simultaneous Protocol for Alexa 488 and Cy5 Fluorochromes;271
12.9.2;9.9.2 Sequential Protocol for Alexa 488, Cy3, and Cy5;272
12.10;9.10 Image Display Window;276
12.11;9.11 Summary;281
12.12;Literature Cited;283
13;Chapter 10: 3D Reconstruction of Confocal Image Data;284
13.1;10.1 Introduction;284
13.2;10.2 Why Reconstruct?;284
13.3;10.3 Defining the 3D Space Using Images;286
13.3.1;10.3.1 Perspective;286
13.3.2;10.3.2 Voxels;287
13.3.3;10.3.3 Resolution;289
13.4;10.4 Types of 3D Reconstruction;291
13.4.1;10.4.1 Maximum Projections;291
13.4.2;10.4.2 Volume Rendering;294
13.4.3;10.4.3 Surface Reconstruction;296
13.5;10.5 The Steps to Reconstruction;299
13.5.1;10.5.1 Planning;299
13.5.2;10.5.2 Acquisition;301
13.5.2.1;10.5.2.1 Deconvolution;301
13.5.3;10.5.3 Alignment;301
13.5.4;10.5.4 Adjustment;302
13.5.4.1;10.5.4.1 Contrast-Limited Adaptive Histogram Equalization (CLAHE);305
13.5.4.2;10.5.4.2 Z-Drop;305
13.5.5;10.5.5 Segmentation;306
13.5.6;10.5.6 Modeling and Visualization;308
13.5.7;10.5.7 Measurement;309
13.6;Literature Cited;312
14;Chapter 11: Analysis of Image Similarity and Relationship;313
14.1;11.1 Introduction;313
14.2;11.2 Colocalization: The Analysis of Similarity in Two Grayscale Images;314
14.3;11.3 Resolution;314
14.4;11.4 Color Perception and Colorimetric Display;316
14.5;11.5 Optimization of Image Quality;318
14.6;11.6 Object-Based Overlap Analysis;319
14.7;11.7 Scatterplot Analysis;322
14.8;11.8 Image Similarity Coefficients;323
14.8.1;11.8.1 Pearson’s Correlation Coefficient;323
14.8.2;11.8.2 Manders’ Overlap Coefficients (MOCs);325
14.8.3;11.8.3 Setting Appropriate and Unbiased Intensity Threshold Level;330
14.8.4;11.8.4 Expanding Correlation Analysis with Spearman’s Rank Correlation Coefficient;331
14.9;11.9 Global Factors Affecting Molecular Clustering;333
14.10;11.10 Take-Home Message;334
14.11;References;335
15;Chapter 12: Ethics and Resources;338
15.1;12.1 Introduction;338
15.2;12.2 Imaging Ethics;339
15.3;12.3 Journal and Office of Research Integrity Guidelines;340
15.4;12.4 Microscopy Society of America Statement on Ethical Digital Imaging;341
15.5;12.5 Available Resources;342
15.6;Additional Literature Cited;344
16;Glossary (Terms Are Defined with Respect to Confocal Imaging);346
17;Index;358
Chapter 1 Introduction and Historical Perspective.- Chapter 2 The Theory of Fluorescence.- Chapter 3 Fluorescence Microscopy.- Chapter 4 Specimen Preparation.- Chapter 5 Labeling Considerations for Confocal Microscopy.- Chapter 6 Digital Imaging.- Chapter 7 Confocal Digital Image Capture.- Chapter 8 Types of Confocal Instruments: Basic Principals and Advantages and Disadvantages.- Chapter 9 Setting the Operating Parameters.- Chapter 10 3D Reconstruction of Confocal Image Data.- Chapter 11 Analysis of Image Similarity and Relationship.- Chapter 12 Ethics and Resources.
