Cell Structure and Function by Microspectrofluorometry provides an overview of the state of knowledge in the study of cellular structure and function using microspectrofluorometry. The book is organized into six parts. Part I begins by tracing the origins of modern fluorescence microscopy and fluorescent probes. Part II discusses methods such as microspectroscopy and flow cytometry; the fluorescence spectroscopy of solutions; and the quantitative implementation of fluorescence resonance energy transfer (FRET) in the light microscope. Part III presents studies on metabolism, including the mechanism of action of xenobiotics; biochemical analysis of unpigmented single cells; and cell-to-cell communication in the endocrine and the exocrine pancreas. Part IV focuses on applications of fluorescent probes. Part V deals with cytometry and cell sorting. It includes studies on principles and characteristics of flow cytometry as a method for studying receptor-mediated endocytosis; and flow cytometric measurements of physiologic cell responses. Part VI on bioluminescence discusses approaches to measuring chemiluminescence or bioluminescence in a single cell and measuring light emitted by living cells.

ContributorsPrefaceTomas Hirschfeld-In MemoriamPart 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 ReferencesPart 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 ReferencesPart 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 ReferencesPart 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 ReferencesPart 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 ReferencesPart 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 ReferencesIndex