Cell Structure and Function by Microspectrofluorometry

Contributors

Preface

Tomas Hirschfeld—In Memoriam

Part I History


1. The Origins of Modern Fluorescence Microscopy and Fluorescent Probes

I. Introduction

II. The First Fluorescence Microscopes

III. Technical Progress

IV. Advances in Biomedical Applications

V. Modern Fluorescence Microscopy in Cell and Molecular Biology

VI. Development of Immunofluorescence

References

Part II Methods


2. Microspectroscopy and Flow Cytometry


3. From Solution Spectroscopy to Image Spectroscopy

I. Fluorescence Spectra

II. Fluorescence Excitation Spectrum

III. Fluorescence Lifetime and Yield

IV. Fluorescence Polarization

References


4. High-Resolution Fluorescence and Phase Microscopy in Conjunction with Micromanipulation for In Situ Study of Metabolism in Living Cells

I. Introduction

II. Resolution of Transmission and Fluorescence Microscopes

III. Microscope Methods

IV. Long-Working-Distance Condenser for Micromanipulation

V. Applications of Spectroscopy to Fluorescence Microscopy

VI. Instrument Design

VII. Application of Photography

VIII. Future Developments

References


5. FRET Microscopy: Digital Imaging of Fluorescence Resonance Energy Transfer. Application in Cell Biology

I. Introduction

II. Theory of Fluorescence Resonance Energy Transfer

III. Measurement of FRET: Data Acquisition and Analysis

IV. Experimental Methods and Results

V. Discussion and Future Prospects

References


6. Fluorescence Scanning Instrumentation

I. Introduction

II. Stage Scanning Microfluorometers

III. Laser Scanning Microfluorometers

IV. Composition of a Laser Scanning Microscope

V. Confocal Laser Scanning

VI. Characteristics of Laser Scanning

VII. Applications in Laser Scanning

References


7. Fluorescence Microscopy in Three Dimensions: Microtomoscopy

I. Introduction

II. Confocal Microscopy

III. Applications

IV. Discussion

References


8. Fluorescence Photochemical Techniques for the Study of Transport in Cytoplasm and Cytoplasmic Models

I. Introduction

II. Apparatus and Methodology

III. Applications to Cytoplasmic Transport

IV. Applications to Cytoplasmic Models In Vitro

V. Fluorescence Photoactivation

VI. Conclusions

References


9. Principles of Frequency-Domain Fluorescence Spectroscopy and Applications to Protein Fluorescence

I. Introduction

II. Comparison of Time and Frequency-Domain Measurements

III. Theory of Frequency-Domain Fluorometry

IV. Tryptophan Fluorescence from Proteins

V. 2-GHz Frequency-Domain Fluorometry

VI. Additional Applications of Frequency-Domain Fluorometry

VII. Future Developments

References


10. The First Picosecond in Vision

I. Introduction

II. Picosecond Time-Resolved Fluorescence Techniques

III. Picosecond Fluorescence Spectroscopy Results

IV. Discussion

References

Part III Metabolism


11. Microspectrofluorometry of Single Living Cells: Quo Vadis

I. Introduction

II. Instrumentation and Methods in Microspectrofluorometry

III. Biological Material

IV. Spatiotemporal Organization of Cell Metabolism

V. Spatiotemporal Mapping of Other Organelles: Lysosomes

VI. Fluorescence Detection of Multiorganelle Complexes Associated with the Cell's Detoxification Function

VII. Other Applications

VIII. Conclusions

References


12. Mechanism of Action of Xenobiotics: from Molecular Spectral Studies to Microspectrofluorometry of Living Cells

I. Introduction

II. Mechanism of Action of Polycyclic Aromatic Hydrocarbons

III. Mechanism of Action of Antipsoriatic Drugs

IV. Anticancer Drugs

V. Conclusions

References


13. Microfluorometry as a Tool for Biochemical Analysis in Unpigmented Single Cells

I. An Example of Convenient Apparatus

II. Resolution of a Complex Cell Fluorescence Spectrum

III. Evaluation of Enzymatic Activities in Intact Living Cells

IV. Use of Fluorescence with Pulsed Excitation

V. General Conclusions

References


14. Fluorescence in the Study of Direct Intercellular Communications: the Case of Pancreatic Cells

I. Introduction

II. Fluorescence Approaches to Direct Intercellular Communications

III. Intercellular Communication Network in the Pancreas

IV. Concluding Remarks

References

Part IV Fluorescent Probes


15. Approaches to the Study of Spatial and Temporal Changes in the Structure and Chemistry of Cells

I. Introduction

II. Approaches to the Study of Cellular Dynamics

III. Experimental Studies

IV. Prospectus

References


16. Fluorescence Studies of Microtubule Dynamics in Living Cells

I. Introduction

II. Microtubule Structure, Intrinsic Polarity, and Organization

III. Spindle Structure and Function

IV. Spindle Lability

V. Fluorescence Approaches to Analyzing Assembly Pathways

VI. Fluorescence Microscopy, Photobleaching, and Digital Image Processing

VII. Microtubule Assembly Occurs by a Dynamic Instability Mechanism

VIII. Comparison with Other Microtubule Arrays

IX. Future Directions

References


17. Optical Measurement of Membrane Potential in Invertebrate Ganglia and Mammalian Cortex

I. Introduction

II. Some Optical Signals are Potential-Dependent

III. Mechanisms

IV. Dyes

V. Recording Activity of Individual Neurons in a Molluscan Central Nervous System

VI. Monitoring Activity in Mammalian Brains

VII. Summary

References


18. Measurement of Free Calcium Concentration inside Single Cells with New Fluorescent Calcium Indicators

I. Introduction

II. Methods

III. Experimental Results

IV. Applications to Cell Systems

V. Future Directions

References

Part V Cytometry and Cell Sorting


19. Flow Cytometric Analysis of Ligand Binding and Endocytosis

I. Introduction

II. Ligand Binding

III. Ligand Internalization

IV. Ligand Acidification

V. Ligand Degradation

VI. Conclusion

References


20. Flow Cytometric Measurements of Physiologic Cell Responses

I. Introduction

II. Physiologic Probes

III. Instrumentation

References


21. Cellular Endogenous Fluorescence: a Basis for Preparing Subpopulations of Functionally Homogeneous Cells

I. Introduction

II. Technique of Autofluorescence-Activated Cell Sorting

III. Purification of Pancreatic B Cells

IV. Functional Heterogeneity in the Pancreatic B-Cell Population

V. Subpopulations Homogeneous in Cellular Hormone Content

VI. Subpopulations Homogeneous in Cellular Glucose Responsiveness

VII. Subpopulations Homogeneous in Sensitivity to Diabetogenic Agents

VIII. Conclusions

References

Part VI Bioluminescence


22. Approaches to the Measurement of Chemiluminescence or Bioluminescence in a Single Cell

I. Introduction

II. Materials and Methods

III. Results and Discussion

References


23. The Measurement of Light Emitted by Living Cells

I. Introduction

II. Electronically Excited States of Molecules

III. Methods for Absolute Calibration and Measurements in Bioluminescence

IV. Methods of Detection of Singlet Oxygen in Biological Reactions by Use of Chemiluminescent Probes

V. The Origin of Bioluminescence

VI. Bioluminescent Systems

VII. Colors of Firefly Bioluminescence

VIII. Experimental Evidence for the Optimization Model of Firefly Fluorescence

IX. Applications of Firefly Bioluminescence to Environmental Photobiology

X. Evolution of Bioluminescence in Bacteria

XI. Emission Spectrum of the Microsomal Chemiluminescence of a Proximate Carcinogen, 7,8-Diol-Benzo(a)Pyrene

XII. Conclusions

References

Index