Fluorescence Microscopy of Living Cells in Culture Part A . Fluorescent Analogs, Labeling Cells, and Basic Microscopy

349 Pages·1988·16.6 MB·English

by  Yu-Li Wang

METHODS IN CELL BIOLOGY
VOLUME 29
Fluorescence Microscopy of Living Cells in Culture
Part A. Fluorescent Analogs, Labeling Cells,
and Basic Microscopy
Series Editor
LESLIE WILSON
Depcirtment of Biological Sciences
Uni\-ersity of Califorriia, Sarita Barbara
Sarifn Barbara, Califorriia
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IASCBI
METHODS IN CELL
BIOLOGY
Prepared under the Auspices of the American Society for Cell Biology
VOLUME 29
Fluorescence Microscopy of Living Cells in Culture
Part A. Fluorescent Analogs, Labeling Cells,
and Basic Microscopy
Edited by
YU-LI WANG
CELL BIOLOGY GROUP
WORCESTER FOUNDATION FOR EXPERIMENTAL BIOLOGY
SHREWSBURY, MASSACHUSETTS
D. LANSING TAYLOR
DEPARTMENT OF BIOLOGICAL SCIENCES
CENTER FOR FLUORESCENCE RESEARCH IN BIOMEDICAL SCIENCES
CARNEGIE-MELIBN UNIVERSITY
PITTSBURGH. PENNSYLVANIA
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CONTENTS
CONTRIBUTORS xi
PREFACE xiii
1. Fluorescent Analog Cytochemistry: Tracing Functional Protein
Components in Living Cells
Yu-li Wang
1. Introduction 1
11. Equipping the Laboratory for Fluorescent Analog Cytochemistry 3
111. Preparation of Fluorescent Analogs 5
IV. Assays of Fluorescent Analogs and Preparations for Microinjection 7
V. Delivery of the Conjugates and Handling of Living Cells 8
VI. Data Collection and Interpretation 9
VII. Prospectus 11
References 12
2. Fluorescent Analogs of kptides and Hormones
Frederick R. MaxJeId
I. Introduction 13
11. Designing a Fluorescent Analog 14
111. Characterization of Fluorescent Analogs 17
IV. Examples of the Use of Fluorescent Analogs
of Hormones and Peptides 21
References 21
3. Fluorescent Analogs of Toxins
Kimon J Angelides
I. Introduction 29
11. Examples of Fluorescent Toxins 31
111. Selection, Preparation, and Utilization of Fluorescent Toxin Analogs 34
IV. Use of Fluorescent Toxin Analogs 48
V. Requirements for Successful Application of Toxin Analogs 56
References 57
V
vi CONTENTS
4. Preparation of Fluorescently Labeled Dextrans and Ficolls
Katherine LubyPhelps
1. Introduction 59
11. Methods 63
111. Further Considerations in the Use of Fluorescent Dextrans and Ficolls 71
References 72
5. A Fluorescent Derivative of Ceramide: Physical Properties
and Use in Studying the Golgi Apparatus of Animal Cells
Richard E. hgano
I. Introduction 75
11. Synthesis and Purification of C,-NBD-ceramide 76
111. Use of Resonance Energy 'Ransfer (RET) to Study Cs-NBD-ceramide
in Liposomes 78
IV. Properties of C,-NBD-ceramide in Cells 82
V. Prospectus 84
References 85
6. Fluorescent Labeling of Cell Surfaces
Michael Midin
1. Introduction 87
11. Direct Chemical Labels of Cell Surfaces 88
111. Fluorescent Ligands as Labels of Cell Surfaces 92
References 101
7. Fluorescent Labeling of Mitochondria
Lan Bo Chen
I. Introduction 103
11. Unique Property of Mitochondria: High Membrane Potential 105
111. Fluorescent Dyes with Delocalized Positive Charges 106
IV. Rhodamine 123 as a Conditional Supravital Dye 107
V. Methods of Labeling and Quantitation 109
VI. Mitochondrial Localization 111
VII. Mitochondrial Morphology 111
VIII. Mitochondrial Motility and Distribution 113
IX. Monitoring Mitochondrial Membrane Potential 115
X. Monitoring Mitochondrial Total Electrochemical Gradient 118
XI. Flow Cytometry, Cell Sorting, and Other Applications 119
References 120
CONTENTS vii
8. Fluorescent Labeling of Endoplasmic Reticulum
Mark Terasaki
I. Introduction 125
11. . Materials 127
111. Labeling Procedures 129
IV. Analysis of the Staining 131
V. Summary 134
References 134
9. Fluorescent Lnbeling of Endocytic Compartments
Joel Swanson
1. Introduction 137
11. Fluorescent Probes 138
111. Identifying Compartments of the Endocytic Pathway 146
References 150
10. Incorporation of Macromolecules into Living Cells
Fbul L. McNeil
1. Introduction 153
11. Strategies for Loading Living Cells 154
111. Selected Techniques for Loading Living Cells 156
IV. Evaluation and Analysis of Loaded Cells 166
References 172
1 1. Hapten-Mediated Immunocytochemistry: The Use of Fluorescent
and Nonfluorescent Haptens for the Study of Cytoskelernl
Dynamics in Living Cells
Gary J Gorbsky and Gary G. Borisy
I. Introduction 175
11. Advantages of Hapten-Mediated Immunocytochemistry 176
111. Preparation of Reagents 178
1V. Application of Hapten-Mediated Immunocytochemistry 181
V. Conclusion and Future Outlook 187
V1. Technical Appendix 188
References 192
...
Vlll CONTENTS
12. Culturing Cells on the Mircroscope Stage
Nancy M. McKenna and Yu-li Wang
I. Introduction 195
11. Requirements of the Microscope Culture System 196
111. Cell Culture Chambers 197
IV. Microscope Incubators 20 1
References 205
13. Basic Fluorescence Microscopy
D Lansing lbylor and E. D. Salmon
I. Introduction 208
11. Microscope Design 215
111. Practical Issues of Epiillumination Fluorescence Microscopy 229
IV. Basic Setup of an Epiillumination Fluorescence Microscope 234
References 236
14. Quantitative Fluorescence Microscopy Using Photomultiplier lhbes
and Imaging Detectors
John E. Wampler and Karl Kutz
I. Introduction 239
11. Accessories for Quantitative Fluorescence Analysis 256
111. Detectors for Quantitative Fluorescence Microscopy 260
References 265
15. Characteristics of Low Light Level Television Cameras
Kenneth R. Spring and R. Joel Lowy
I. Introduction 270
11. Requirements for a Low Light Level Video Camera 270
111. Criteria for Camera Selection and Evaluation 282
IV. Performance Characteristics of Existing Low Light Level Cameras 285
V. Future Developments 288
References 289
16. Solid-state Imagers for Microscopy
R. S. Aikens, D A. Agard, and 1 W Sedat
I. The History of Solid-State Imagers 292
11. Theory of CCD Imagers 293
111. CCD Camera Implementations 300
ix
CONTENTS
1V. CCD Performance 302
V. Quantitative Imaging with CCDs 309
VI. Future Trends in Solid-state Detector Development 312
VII. Summary 313
References 313
INDEX 315
CONTENOTFS RECENTV OLUMES 329