Methods in Cell Biology

Transcription

1 Methods in Cell Biology VOLUME 7 0 Cell Biological Applications of Confocal Microscop y Second Edition Brian Matsumoto

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3 Contributors Preface ix x i 1. Introduction to Confocal Microscopy Shirley]. Wright and David J. Wright I. Introduction 2 II. Principle of Confocal Microscopy 7 III. Major Components of a Confocal Microscope 1 4 IV. Designs of Confocal Microscopes 2 3 V Choosing and Setting Up a Confocal Microscope System 4 9 VI. Specimen Preparation 5 2 VII. Applications of Confocal Microscopy 5 2 VIII. Alternatives to Confocal Microscopy 6 8 IX. Conclusions and Future Directions 7 3 X. Resources for Confocal Microscopy 7 4 References Direct-View High-Speed Confocal Scanner: The CSU-1 0 Shinya Inoue and Ted Inou e I. Introduction 8 8 II. Overview of Confocal Microscopes 9 0 III. Design of the CSU IV. Sample Biological Applications 9 3 V. Low Bleaching of Fluorescence Observed with the CSU VI. Very-High-Speed Full-Frame Confocal Imagin g with the CSU VII. Haze Removal, Image Sharpening, and Dynamic Stereo Image Generatio n by Digital Signal Processing 10 5 VIII. Mechanical and Optical Performance of the CSU IX. Potentials of the CSU X. Addendum A (April, 2002) 122 XI. Addendum B (personal communication from Dr. Kenneth R. Spring, June, 2002) 124 References 125

4 3. Introduction to Multiphoton Excitation Imaging for the Biological Sciences Victoria E. Centonze I. Introduction 129 II. Principles of Multiphoton Imaging 13 1 III. Advantages of Multiphoton Imaging 13 3 IV. Imaging Parameters 137 V. Applications of Multiphoton Imaging 14 1 VI. New Development in Nonlinear Optics 14 4 VII. Conclusion 14 5 References Confocal Microscopy : Important Considerations for Accurate Imagin g Lars Majlof and Per-01a Forsgren I. Introduction 14 9 II. Factors Affecting Confocal Imaging 15 0 III. Conclusions 16 3 References Multicolor Laser Scanning Confocal Immunofluorescence Microscopy : Practical Application and Limitation s T. Clark Brelje, Martin W. Wessendorf, and Robert L. Sorenson I. Introduction 16 6 II. Immunofluorescence Histochemistry 16 7 III. Instrumentation 18 5 IV Approaches to Multicolor Laser Scanning Confocal Microscopy 19 1 V. Practical Aspects of Multicolor Laser Scanning Confocal Microscopy 20 7 VI. Multicolor Laser Scanning Confocal Microscopy Considerations 23 3 VII. Conclusions 240 References Practical Aspects of Objective Lens Selection for Confocal and Multiphoto n Digital Imaging Technique s Gerald S. Benha m I. Introduction 247 II. Confocal Microscopy Resolution 24 9 III. Infinity versus Finite Lens Systems 254 IV. Objective Lens Nomenclature and Features 25 6 V. Objective Lens Aberrations 26 1 VI. Dry (Nonimmersion) and Immersion Objective Lenses 26 3 VII. Epi (Reflected Light) Objectives 26 6 VIII. Live Cell and Fixed Tissue Confocal Applications and Objective Lens Selection 26 8 IX. Possible Solutions for Potential Problematic Applications 27 8 X. Optimum Objective Lenses for Multiphoton Applications 28 8 XI. Transmission Data for Confocal and Multiphoton Objectives 28 9 XII. Conclusions 29 0 References 293

5 7. Resolution of Subcellular Detail in Thick Tissue Sections : Immunohistochemical Preparation and Fluorescence Confocal Microscop y Irene L. Hale and Brian Matsumoto I. Introduction: Development of Fluorescence Microscopy Technique s for Resolution of Subcellular Detail 302 II. Immunohistochemical Preparation of Thick Tissue Sections 308 III. Confocal Imaging Parameters for Maximum Resolution 32 1 IV. Applications 326 V. Appendix: Procedures for Immunohistochemical Preparation 329 References Confocal Fluorescence Microscopy for Tetrahymena thermophil a E. S. Cole, K. R. Stuart, T. C. Marsh, K. Aufderheide, and W. Ringlien I. Introduction 338 II. Indirect Immunofluorescence and Confocal Microscopy 338 III. Fluorescence in Situ Hybridization Detection of Tetrahymena thermophila rdna for Confocal Microscopy 344 IV. Live Fluorescence Microscopy 347 References Confocal Imaging of Drosophila Embryos Stephen W. Paddock I. Introduction 36 1 II. Preparation of the Embryos 36 3 III. Imaging Embryos 36 5 IV. Image Presentation 37 0 V. Multiple Labels 37 2 VI. Conclusions 37 5 References Confocal Fluorescence Microscopy of the Cytoskeleton of Amphibia n Oocytes and Embryos David L. Gard I. Introduction 38 0 II. Fixation of Xenopus Oocytes and Eggs for Confocal Fluorescenc e Microscopy of the Cytoskeleton 38 3 III. Processing Xenopus Oocytes and Eggs for "Whole-Mount " Confocal Microscopy 39 0 IV. An Introduction to Confocal Microscopy and 3-D Reconstruction o f the Cytoskeleton of Xenopus Oocytes 39 7 V. Conclusions 40 8 VI. Recipes and Reagents 40 9 VII. Cytoskeletal and Nuclear Probes for Confocal Microscopy of Xenopus Oocytes 41 0 References 413

6 11. Confocal Fluorescence Microscopy Measurements of ph and Calciu m in Living Cell s Kay-Pong Yip and Ira Kurtz 1. Introduction 41 7 II. Measurement of phi with BCECF 41 8 IIl. Design of a Dual-Excitation Laser Scanning Confocal Fluorescenc e Microscope for Measuring phi with BCECF 42 0 IV. Measurement of phi in the Cortical Collecting Tubule 422 V. Measurement of Intracellular Ca2+ in the Perfused Afferent Arteriole 42 4 VI. Future Development 426 References Confocal and Nonlinear Optical Imaging of Potentiometric Dye s Leslie M. Loew, Paul Campagnola, Aaron Lewis, and Joseph P Wuskel l I. Introduction Overview of Fluorescent Methods for Measuring Membrane Potential 430 IlI. Application of Laser Scanning Microscopy to Quantitative Imaging of Membrane Potential 43 4 IV. Perspectives 44 8 References Measurement of Intracellular Ca2+ Concentration Nicolas Dernaurex, Serge Arnaudeau, and Michal Opa s I. Introduction 45 3 II. Ratio Measurement of pcai with Visible-Light [Ca Z+] Indicators 45 5 IlI. Measurement of Organellar pcai with Genetically Engineered [Ca 2+] Indicators 46 0 References Running and Setting Up a Confocal Microscope Core Facilit y Susan DeMaggi o I. Introduction 47 5 II. Planning Your Facility 47 6 III. Scheduling 47 7 IV. Training Operators and Users 47 8 V. Instrument Care 48 1 VI. Data Storage, Analysis, and Handling 48 2 VII. Core Issues 48 3 VIII. Ancillary Services 485 IX. Resources 48 5 X. Summary 48 5 Index 48 7 Volumes in Series 501