Methods in Cell Biology

VOLUME 7 0

Cell Biological Applications of Confocal Microscopy

Second Edition

Brian Matsumoto

Contributors

ix

Preface

x i

1 . Introduction to Confocal Microscopy

Shirley]. Wright and David J. Wright

I. Introduction

2II. Principle of Confocal Microscopy

7III. Major Components of a Confocal Microscope

1 4IV. Designs of Confocal Microscopes

2 3V Choosing and Setting Up a Confocal Microscope System

4 9VI. Specimen Preparation

5 2VII. Applications of Confocal Microscopy

5 2VIII. Alternatives to Confocal Microscopy

6 8IX. Conclusions and Future Directions

7 3X. Resources for Confocal Microscopy

7 4References

8 1

2 . Direct-View High-Speed Confocal Scanner : The CSU-1 0

Shinya Inoue and Ted Inou e

I. Introduction

8 8II. Overview of Confocal Microscopes

9 0III. Design of the CSU-10

9 1IV. Sample Biological Applications

9 3V. Low Bleaching of Fluorescence Observed with the CSU-10

10 1VI. Very-High-Speed Full-Frame Confocal Imagin g

with the CSU-10

104VII. Haze Removal, Image Sharpening, and Dynamic Stereo Image Generatio n

by Digital Signal Processing

10 5VIII. Mechanical and Optical Performance of the CSU-10

11 7IX. Potentials of the CSU-10

11 8X. Addendum A (April, 2002)

122XI. Addendum B (personal communication from Dr . Kenneth R . Spring ,

June, 2002)

124References

125

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

14 5

4 . 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

16 4

5 . 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

24 1

6 . 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

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 sfor Resolution of Subcellular Detail

302II. Immunohistochemical Preparation of Thick Tissue Sections

308III. Confocal Imaging Parameters for Maximum Resolution

32 1IV. Applications

326V. Appendix: Procedures for Immunohistochemical Preparation

329References

332

8 . Confocal Fluorescence Microscopy for Tetrahymena thermophil a

E . S. Cole, K. R. Stuart, T. C. Marsh, K. Aufderheide, and W. Ringlien

I. Introduction

338II. Indirect Immunofluorescence and Confocal Microscopy

338III. Fluorescence in Situ Hybridization Detection of Tetrahymena thermophila

rDNA for Confocal Microscopy

344IV. Live Fluorescence Microscopy

347References

35 8

9 . Confocal Imaging of Drosophila Embryos

Stephen W. Paddock

I. Introduction

36 1II. Preparation of the Embryos

36 3III. Imaging Embryos

36 5

IV. Image Presentation

37 0V. Multiple Labels

37 2VI. Conclusions

37 5References

37 6

10 . Confocal Fluorescence Microscopy of the Cytoskeleton of Amphibia n

Oocytes and Embryos

David L. Gard

I. Introduction

38 0II. Fixation of Xenopus Oocytes and Eggs for Confocal Fluorescenc e

Microscopy of the Cytoskeleton

38 3III. Processing Xenopus Oocytes and Eggs for "Whole-Mount " Confocal Microscopy 39 0IV. An Introduction to Confocal Microscopy and 3-D Reconstruction o f

the Cytoskeleton ofXenopus Oocytes

39 7V. Conclusions

40 8VI. Recipes and Reagents

40 9VII. Cytoskeletal and Nuclear Probes for Confocal Microscopy of Xenopus Oocytes

41 0References

413

11 . Confocal Fluorescence Microscopy Measurements of pH and Calciu m

in Living Cell s

Kay-Pong Yip and Ira Kurtz

1 . Introduction

41 7II . 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

426

12 . Confocal and Nonlinear Optical Imaging of Potentiometric Dye s

Leslie M. Loew, Paul Campagnola, Aaron Lewis, and Joseph P Wuskel l

I . Introduction

4 2911 . Overview of Fluorescent Methods for Measuring Membrane Potential

430

IlI . Application of Laser Scanning Microscopy to Quantitative Imagingof Membrane Potential

43 4

IV. Perspectives

44 8

References

44 9

13 . 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 [Ca2+ ] Indicators 46 0

References

47 0

14 . 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 7IV. 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

487

Volumes in Series

501