Elsevier S & T

Cell Biological Applications of Confocal Microscopy

Matsumoto, Brian

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Table of contents

Cover
Contentsv
Contributorsix
Prefacexi
Chapter 1. Introduction to Confocal Microscopy1
I. Introduction2
II. Principle of Confocal Microscopy7
III. Major Components of a Confocal microscope14
IV. Designs of Confocal Microscopes23
V. Choosing and Setting Up a Confocal Microscope System49
VI. Specimen Preparation52
VII. Applications of Confocal Microscopy52
VIII. Alternatives to Confocal Microscopy68
IX. Conclusions and Future Directions73
X. Resources for Confocal Microscopy74
References81
Chapter 2. Direct-View High-Speed Confocal Scanner: The CSU-1087
I. Introduction88
II. Overview of Confocal Microscopes90
III. Design of the CSU-1091
IV. Sample Biological Applications93
V. Low Bleaching of Fluorescence Observed with the CSU-10101
VI. Very-High-Speed Full-Frame Confocal Imaging with the CSU-10104
VII. Haze Removal, Image Sharpening, and Dynamic Stereo Image Generation by Digital Signal Processin105
VIII. Mechanical and Optical Performance of the CSU-10117
IX. Potentials of the CSU-10118
X. Addendum A (April, 2002)122
XI. Addendum B (personal communication from Dr. Kenneth R. Spring, June, 2002)124
References125
Chapter 3. Introduction to Multiphoton Excitation Imaging for the Biological Sciences129
I. Introduction129
II. Principles of Multiphoton Imaging131
III. Advantages of Multiphoton Imaging133
IV. Imaging Parameters137
V. Applications of Multiphoton Imaging141
VI. New Development in Nonlinear Optics144
VII. Conclusion145
References145
Chapter 4. Confocal Microscopy: Important Considerations for Accurate Imaging149
I. Introduction149
II. Factors Affecting Confocal Imaging150
III. Conclusions163
References164
Chapter 5. Multicolor Laser Scanning Confocal Immunofluorescence Microscopy: Practical Application a165
I. Introduction166
II. Immunofluorescence Histochemistry167
III. Instrumentation185
IV. Approaches to Multicolor Laser Scanning Confocal Microscopy191
V. Practical Aspects of Multicolor Laser Scanning Confocal Microscopy207
VI. Multicolor Laser Scanning Confocal Microscopy Considerations233
VII. Conclusions240
References241
Chapter 6. Practical Aspects of Objective Lens Selection for Confocal and Multiphoton Digital Imagin245
I. Introduction247
II. Confocal Microscopy Resolution249
III. Infinity versus Finite Lens Systems254
IV. Objective Lens Nomenclature and Features256
V. Objective Lens Aberrations261
VI. Dry (Nonimmersion) and Immersion Objective Lenses263
VII. Epi (Reflected Light) Objectives266
VIII. Live Cell and Fixed Tissue Confocal Applications and Objective Lens Selection268
IX. Possible Solutions for Potential Problematic Applications278
X. Optimum Objective Lenses for Multiphoton Applications288
XI. Transmission Data for Confocal and Multiphoton Objectives289
XII. Conclusions290
References293
Chapter 7. Resolution of Subcellular Detail in Thick Tissue Sections: Immunohistochemical Preparatio301
I. Introduction: Development of Fluorescence Microscopy Techniques for Resolution of Subcellular Det302
II. Immunohistochemical Preparation of Thick Tissue Sections308
III. Confocal Imaging Parameters for Maximum Resolution321
IV. Applications326
V. Appendix: Procedures for Immunohistochemical Preparation329
References332
Chapter 8. Confocal Fluorescence Microscopy for Tetrahymena thermophila337
I. Introduction338
II. Indirect Immunofluorescence and Confocal Microscopy338
III. Fluorescence in Situ Hybridization Detection of Tetrahymena thermophila rDNA for Confocal Micro344
IV. Live Fluorescence Microscopy347
References358
Chapter 9. Confocal Imaging of Drosophila Embryos361
I. Introduction361
II. Preparation of the Embryos363
III. Imaging Embryos365
IV. Image Presentation370
V. Multiple Labels372
VI. Conclusions375
References376
Chapter 10. Confocal Fluorescence Microscopy of the Cytoskeleton of Amphibian Oocytes and Embryos379
I. Introduction380
II. Fixation of Xenopus Oocytes and Eggs for Confocal Fluorescence Microscopy of the Cytoskeleton383
III. Processing Xenopus Oocytes and Eggs for Whole-MountŽ Confocal Microscopy390
IV. An Introduction to Confocal Microscopy and 3-D Reconstruction of the Cytoskeleton of Xenopus Ooc397
V. Conclusions408
VI. Recipes and Reagents409
VII. Cytoskeletal and Nuclear Probes for Confocal Microscopy of Xenopus Oocytes410
References413
Chapter 11. Confocal Fluorescence Microscopy Measurements of pH and Calcium in Living Cells417
I. Introduction417
II. Measurement of pHi with BCECF418
III. Design of a Dual-Excitation Laser Scanning Confocal Fluorescence Microscope for Measuring pHi w420
IV. Measurement of pHi in the Cortical Collecting Tubule422
V. Measurement of Intracellular Ca2+ in the Perfused Afferent Arteriole423
VI. Future Development425
References426
Chapter 12. Confocal and Nonlinear Optical Imaging of Potentiometric Dyes429
I. Introduction429
II. Overview of Fluorescent Methods for Measuring Membrane Potential430
III. Application of Laser Scanning Microscopy to Quantitative Imaging of Membrane Potential434
IV. Perspectives448
References449
Chapter 13. Measurement of Intracellular Ca2+ Concentration453
I. Introduction453
II. Ratio Measurement of pCai with Visible-Light [Ca2+] Indicators455
III. Measurement of Organellar pCai with Genetically Engineered [Ca2+] Indicators460
References470
Chapter 14. Running and Setting Up a Confocal Microscope Core Facility475
I. Introduction475
II. Planning Your Facility476
III. Scheduling477
IV. Training Operators and Users478
V. Instrument Care481
VI. Data Storage, Analysis, and Handling482
VII. Core Issues483
VIII. Ancillary Services485
IX. Resources485
X. Summary485
Index487
Volumes in Series501

Book details

Vendor Elsevier S & T
SKU 9780124802773
ISBN-13 9780080496580
Author Matsumoto, Brian
Edition 2nd
Category Science
Subject Microscopes & Microscopy

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This volume of the acclaimed Methods in Cell Biology series provides specific examples of applications of confocal microscopy to cell biological problems. It is an essential guide for students and scientists in cell biology, neuroscience, and many other areas of biological and biomedical research, as well as research directors and technical staff of microscopy and imaging facilities.

An integrated and up-to-date coverage on the many various techniques and uses of the confocal microscope (CM).

Key Features
* Includes detailed protocols accessible to new users
* Details how to set up and run a "Confocal Microscope Core Facility"
* Contains over 170 figures

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