© 2000 • 838 pp., illus., appendices, index
Plastic comb binding • ISBN  0-87969-541-2

This title also available in: Cloth

In the past decade, advances in microscopy have been coupled with new methods of culturing and labeling cells to generate the new science of imaging. Imaging technologies allow investigators to look directly inside living cells and probe their form and function in unprecedented detail. This approach is revolutionizing many aspects of biomedical research, particularly neuroscience, in which visual techniques have traditionally been so important.

This manual is the first comprehensive description of the range of imaging technologies being applied to living cells. With its origins in a laboratory course taught at Cold Spring Harbor Laboratory by the editors and contributors, it is packed with the kind of technical detail and practical advice that are essential for success, yet seldom found in the research literature. It covers both established methods and cutting-edge techniques such as multi-photon excitation microscopy and imaging of genetically engineered probes.

Although it is neurons to which these technologies are most commonly applied, the methods described are readily adaptable to many other cell types. This book will therefore be an invaluable aid to investigators in cell and developmental biology and immunology as well as neuroscience who wish to take advantage of the extraordinary insights into cellular function offered by imaging technologies.

  "I think it is safe to say there is something for everyone in this book, at least with respect to light microscopy of fluorescent molecules, and it provides an excellent overview of modern techniques to study structural/functional relationships in living cells. The blend of theoretical chapters with practical step-by-step techniques will ensure that it finds a place on the bookshelves of many cell biologists, especially those interested in excitable cells. The book is not for the novice."
      —Microscopy Today

  "This manual provides a very important addition to the library of anyone whose work, either directly or indirectly, involves neuronal imaging."
      —Trends in Cell Biology

Foreword

Preface

Acknowledgments

Abbreviations

SECTION 1
Microscopes, Image Acquisition, and the Basics of Fluorescence Imaging

1. Microscopy and Microscope Optical Systems (F. Lanni and H.E. Keller)
2. Arc Lamps and Monochromators for Fluorescence Microscopy (R. Uhl)
3. The Use of Liquid-crystal Tunable Filters for Fluorescence Imaging (K.R. Spring)
4. Acousto-optic Tunable Filters for Microscopy (E.S. Wachman)
5. Video Microscopy, Video Cameras, and Image Enhancement
   Reprint (modified) from Cells: A Laboratory Manual
6. The Application of Scientific-grade CCD Cameras to Biological Imaging (M. Christenson)
7. Infrared Videomicroscopy (H.-U. Dodt, G. D'Arcangelo, and W. Zieglgänsberger)
8. Grating Image Systems for Optical Sectioning Fluorescence Microscopy of Cells, Tissues, and Small Organisms (F. Lanni and T. Wilson)
9. Analysis of Dynamic Optical Imaging Data (P.P. Mitra, B. Pesaran, and D. Kleinfeld)
10. Maintaining Live Cells and Tissue Slices in the Imaging Setup (M.E. Dailey)

SECTION 2
Confocal Microscopy

11. Confocal Microscopy: Principles and Practice (A. Fine)
12. Practical Limits to Resolution in Fluorescence Light Microscopy (E.H.K. Stelzer)
13. In Vivo Imaging of CNS Neuron Development (H.T. Cline, J.E. Edwards, I. Rajan, G.-Y. Wu, and D.-J. Zou)
14. The Zebrafish Hindbrain: A Transparent System for Imaging Motor Circuitry (D.M. O'Malley and J.R. Fetcho)
15. Fast Confocal Fluorescence Imaging of Subcellular Calcium Dynamics (J. Eilers)
16. Imaging Calcium Sparks in Excitable Cells (M.B. Cannell and C. Soeller)

SECTION 3
Multiphoton Microscopy

17. Principles of Multiphoton-excitation Fluorescence Microscopy (W. Denk)
18. Lasers for Multiphoton Microscopy (F.W. Wise)
19. Two-photon Cross Sections of Indicators (C. Xu)
20. Two-photon Microscopy for 4D Imaging of Living Neurons (S.M. Potter)
21. Multiphoton Imaging of Embryonic Development (W.A. Mohler and J.M. Squirrell)
22. Two-photon Imaging of Neuronal Function in the Neocortex In Vivo (K. Svoboda, D.W. Tank, R.A. Stepnoski, and W. Denk)
23. Two-photon Imaging of Neocortical Microcirculation (D. Kleinfeld and W. Denk)

SECTION 4
Photoactivation

24. Basics of Photoactivation (G.C.R. Ellis-Davies)
25. Design and Application of Caged Neurotransmitters (G.P. Hess)
26. Uncaging Calcium in Neurons (K.R. Delaney)
27. Photostimulation with Caged Glutamate (K. Kandler, R.S. Givens, and L.C. Katz)
28. Infrared-guided Photostimulation of Neurons in Brain Slices (H.-U. Dodt, A. Frick, and W. Zieglgänsberger)
29. Spatially Resolved Flash Photolysis Via Chemical Two-photon Uncaging (G.J. Augustine, D.L. Pettit, and S.S.-H. Wang)

SECTION 5
Calcium Imaging

30. How Calcium Indicators Work (S.R. Adams)
31. Some Quantitative Aspects of Calcium Fluorimetry (E. Neher)
32. Calibration of Fluorescent Calcium Indicators (F. Helmchen)
33. A Single-compartment Model of Calcium Dynamics in Nerve Terminals and Dendrites (F. Helmchen and D.W. Tank)
34. Loading Brain Slices with AM Esters of Calcium Indicators (R. Yuste)
35. Dye Loading with Patch Pipets (J. Eilers and A. Konnerth)
36. Measuring Calcium Fluxes in Dendritic Domains of Neurons in Brain Slices (O. Garaschuk and A. Konnerth)
37. Monitoring Presynaptic Calcium Dynamics with Membrane-permeant Indicators (W.G. Regehr)
38. Imaging Intracellular Calcium-concentration Microdomains at a Chemical Synapse (R. Llinás and M. Sugimori)
39. Monitoring Intramitochondrial Calcium with Rhod-2 (M. Hoth and R.S. Lewis)
40. Generation of Controlled Calcium Oscillations in Nonexcitable Cells (R.E. Dolmetsch and R.S. Lewis)
41. Calcium Imaging of Retinal Activity (R.O.L. Wong)
42. Calcium Imaging of Identified Astrocytes in Hippocampal Slices (J. Kang and M. Nedergaard)
43. High-speed Calcium Imaging of Neurons in Brain Slices (W.N. Ross, R. Gray, and N. Lasser-Ross)
44. In Vivo Calcium Imaging in Blowfly CNS (A. Borst and S. Single)

SECTION 6
Imaging Other Aspects of Neuronal Function

45. Intrinsic Signal Imaging in the Neocortex (A. Grinvald, A. Shmuel, I. Vanzetta, E. Shtoyerman, D. Shoham, and A. Arieli)
46. Intrinsic Signal Optical Imaging as a Tool to Visualize the Development of Functional Maps in the Mammalian Visual Cortex (T. Bonhoeffer)
47. Intrinsic Signal Optical Imaging in Brain Slices (B.A. MacVicar)
48. Rapid Changes in Light Scattering Associated with Secretion from Peptidergic Nerve Terminals (B.M. Salzberg, M. Muschol, and A.L. Obaid)
49. Imaging with Voltage-sensitive Dyes: Spike Signals, Population Signals, and Retrograde Transport (J.-Y. Wu, L.B. Cohen, Y. Tsau, Y.-W. Lam, M. Zochowski, and C.X. Falk)
50. Voltage-sensitive-dye Imaging in the Neocortex: Visualization of Coherent Neuronal Assemblies (A. Grinvald, R. Hildesheim, D. Shoham, D.E. Glaser, and A. Arieli)
51. Imaging Membrane-potential Changes with Intracellular Voltage-sensitive Dyes (S. Antic and D. Zecevic)
52. Imaging Sodium Dynamics in Neurons (W.N. Ross, J.C. Callaway, and N. Lasser-Ross)
53. Imaging Synaptic Vesicle Dynamics at the Frog Neuromuscular Junction with the Styryl Dye FM1-43 (U. Becherer, J.K. Angleson, and W.J. Betz)
54. Imaging Exocytosis with Total Internal Reflection Microscopy (J.A. Steyer and W. Almers)

SECTION 7
Genetically Engineered Fluorescent Probes

55. Physiological Indicators Based on Fluorescence Resonance Energy Transfer (R.Y. Tsien)
56. Cellular Imaging of Bioluminescence (J.D. Plautz and S.A. Kay)
57. Green Fluorescent Proteins for Measuring Signal Transduction: A Voltage-sensor Prototype (M.S. Siegel and E.Y. Isacoff)
58. Introduction of Green Fluorescent Protein into Hippocampal Neurons through Viral Infection (R. Malinow, Y. Hayashi, M. Maletic-Savatic, S.H. Zaman, J.-C. Poncer, S.-H. Shi, and J.A. Esteban)
59. Imaging Exocytosis with pH-sensitive Green Fluorescent Proteins (G. Miesenböck)
60. Targeted Recombinant Aequorins: New Tools in the Study of Calcium Homeostasis (T. Pozzan and R. Rizzuto)

APPENDICES

Appendix 1: Glossary

Appendix 2: Electromagnetic Spectrum

Appendix 3: Microscopy: Lenses, Filters, and Emission/Excitation Spectra

Appendix 4: Cautions

Appendix 5:

A. Sample Reagents

B. Sample Protocols for Brain Slice Preparation and Neuronal Culture

Appendix 6: How to Calculate and Prepare Calcium-EGTA Buffers

Appendix 7: Manufacturers’ and Suppliers’ Address List

Appendix 8: Trademarks

Index