Modern Fluorescence Spectroscopy

Since the appearance of the first two volumes of Modern Fluorescence Spectroscopy in 1976, important advances continue to be made in both the techniques and applications of molecular luminescence. In terms of "hardware," it is only recently that the application of laser excitation to molecular fluorometry has become feasible under conditions that are analy­ tically realistic. The improvements that can be effected in sensitivity, analy­ tical selectivity, and ability to handle "difficult" samples by laser fluorometry have only begun to be exploited. Likewise, time-resolved fluorometry has received widespread use in fundamental studies (a sizable number of which deal with biological systems), but has as of yet received relatively little analytical utilization. The use of electronic array detectors offers the promise of obtaining luminescence spectra more rapidly, and perhaps ultimately with greater sensitivity, than is possible by the use of scanning instruments equipped with conventional detectors. The increasing capabilities of microcomputers and the increasing sophistication of "smart" spectroscopic instrumentation signify that much more efficient acquisition and use can now be achieved of the information contained in the "excitation-emission matrix" inherent in the luminescence phenomenon.

1. Structural Interpretation of Fluorescence Spectra by Automated File Searching. Implementation and Applications in Liquid Chromatography.- A. Introduction.- B. Alternatives for Computerized Spectral Interpretation.- 1. File Searching.- 2. Pattern Recognition.- 3. Artificial Intelligence.- 4. Choosing an Approach for Fluorescence Data.- C. An Initial Approach.- 1. The Library and Software Structure.- 2. Search Procedure.- 3. Index of Dissimilarity.- 4. Uniqueness of Spectra.- 5. Performance with Data from the Laboratory.- D. Using More Subtle Features.- 1. Compression of Data.- 2. Search Procedure.- 3. Further Compression of Library Size.- 4. Performance.- E. Limitations.- F. Hardware for an LC/Fluorescence System.- 1. Ideal Components.- 2. A Practical Solution.- 3. Problems.- G. Reverse Searching.- H. Performance and Outlook.- References.- 2. Fluorescence Detection in Liquid and Gas Chromatography: Techniques, Examples, and Prospects.- A. Introduction.- B. Detection of Compounds That Exhibit Significant Native Fluorescence.- 1. Description of a Typical Analysis Using HPLC with Fluorescence Detection.- a. Sample Cleanup.- b. Chromatographic Separation.- c. Fluorescence Detection.- 2. Enhancement of the Sensitivity of HPLC/Fluorescence Analytical Procedure.- a. Composition of the Mobile Phase.- b. The Effect of Dissolved Gases.- 3. Acquisition of Fluorescence Spectra of HPLC Eluants.- a. Stopping the Flow of the Chromatograph and Recording the Spectrum.- b. Rapid Spectral Scanning while the Chromatograph Continues to Deliver Mobile Phase.- c. Rapid Spectral Acquisition with Electronic Array Detectors.- C. Detection of Compounds That Do Not Exhibit a Significant Fluorescence Quantum Yield.- 1. Pre-Column Derivatization.- 2. Post-Column Derivatization.- D. Further Instrumental Considerations in HPLC Detection by Fluorescence.- 1. Flow Cells and Connections to the Column.- 2. Illumination Sources.- E. Future Trends in Fluorometric HPLC Detection.- F. Fluorescence Detection in Gas Chromatography.- 1. Effluent Trapping Procedures.- 2. Measurement of Vapor-Phase Fluorescence Spectra.- 3. Matrix Isolation Spectroscopy.- References.- 3. Reaction Rate Methods in Fluorescence Analysis.- A. Introduction.- B. Characteristics of Fluorescence Kinetic Measurements.- 1. General Terminology and Instrumental Considerations.- 2. General Advantages and Limitations of Kinetic Methods.- 3. Advantages and Limitations of Fluorometric Kinetic Methods.- 4. Precision and Signal-to-Noise Ratio Considerations.- 5. Optimization of Fluorometric Kinetic-Based Procedures.- C. Applications of Fluorometric Kinetic Methods.- 1. Enzymatic Methods.- a. Determination of Enzymes.- b. Determination of Substrates.- c. Other Enzymatic Determinations.- 2. Catalytic Kinetic Methods.- 3. Noncatalytic Methods.- a. Determination of Metals.- b. Determination of Organic Compounds.- D. Instrumentation for Fluorescence Rate Measurements.- 1. Introduction.- 2. Photon Counting.- 3. Rapid-Scanning Devices.- 4. Microprocessors.- 5. Immobilized Enzymes.- E. Conclusions.- References.- 4. Principles and Practice of Fluoroimmunoassay Procedures.- A. Introduction.- 1. The Antibody Response.- 2. Antibody Structure.- 3. The Antigen-Antibody Binding Reaction.- 4. Antibody as an Analytical Reagent.- B. Principles of Immunoassay.- 1. Employing Labeled Antigen.- 2. Employing Labeled Antibody.- 3. Choice of Label.- C. Fluoroimmunoassay Reagents and Equipment.- 1. Antiserum.- a. Anti-Protein Serum.- b. Anti-Hapten Serum.- 2. Label.- 3. Labeled Reagents.- a. Labeled Protein Antigens.- b. Labeled Haptens.- c. Labeled Antibodies.- 4. Solid Phases.- 5. Instrumentation.- D. Fluoroimmunoassay Techniques.- 1. Procedures that Employ Labeled Antigen.- a. Separation FIA.- b. Nonseparation FIA.- i. Quenching FIA.- ii. Enhancement FIA.- iii. Polarization FIA.- iv. Fluorescent Excitation Transfer Immunoassay.- v. Substrate-Labeled FIA.- vi. Indirect Quenching FIA.- 2. Procedures That Employ Labeled Antibody.- a. Separation IFMA.- b. Nonseparation IFMA.- E. Fluoroimmunoassay Limitations and Interfering Factors.- 1. Sensitivity of Fluorometry.- 2. Sample Interferences.- a. Nonspecific Binding of Fluorescein-Labeled Reagents.- b. Spectroscopic Factors.- F. Selected Examples of Fluoroimmunoassay Development and Application.- 1. Nonseparation FIA of Haptens.- a. Quenching FIA.- b. Polarization FIA.- 2. Nonseparation FIA of a Protein.- 3. Separation FIA of a Hapten.- G. Future Trends and Developments.- 1. Basic Instrumentation.- 2. Assay Reagents.- 3. Assay Techniques.- 4. Sensitivity Improvement.- a. Multifluorophore Labeling.- b. Background Rejection.- i. Bleaching Lifetime Discrimination.- ii. Fluorescence Lifetime Discrimination.- H. Summary.- I. Appendix: Glossary of Terms.- References.- 5. Fluorometric Studies of Biologically Important Molecular Complexes.- A. Introduction.- B. Complexes of Acridine Drugs with Nucleotides and DNA.- 1. Complexes with Nucleotides.- 2. Complexes with DNA.- 3. Criteria for the Accurate Determination of Binding Parameters.- 4. Cytogenetical and Medical Applications.- a. The Staining of Human Metaphase Chromosomes.- b. Phototherapy and Photoprotection.- C. Complexes Involving Hydrogen Bonds.- 1. The Importance of the Franck-Condon Principle in Interpreting Spectral Shifts.- 2. Examples of Hydrogen Bonding and Excited-State Proton Transfer.- D. Complexes between Oligopeptides and Proteins with Nucleic Acids.- 1. Complexes Involving Free Tryptophan or Oligopeptides.- 2. Complexes Involving Proteins.- 3. Mechanisms of Fluorescence Quenching and Binding.- 4. Photosensitized Splitting of Thymine Dimers by Proteins.- E. Dynamics of Membranes Probed by Fluorophores Forming Complexes.- 1. Intermolecular Excimer Formation.- 2. Intramolecular Excimer Formation.- 3. Fluorophore-Polar Group Ground-State Complex Formation.- F. Intrinsic and Extrinsic Fluorescent Probes of Biomolecules.- 1. Nanosecond Fluorescence Polarization Studies.- 2. Excitation Energy Transfer Studies.- 3. Excited States of Nucleic Acids.- 4. Fluorometric Techniques in Photosynthesis.- 5. Circular Polarization of Fluorescence of Biomolecules.- References.- 6. Fluorometric Quantification of Specific Chemical Species in Single Cells.- A. Introduction.- B. Instrumentation and Methods.- 1. Standard Microtechniques.- 2. Microfluorophotometers.- 3. Optical Multichannel Analyzers.- 4. Flow Microfluorometers.- 5. Dual Laser Flow Cytometers.- C. Analytical Considerations.- 1. Detection Limits.- 2. Standardization.- 3. Errors in Measurement.- 4. Coefficient of Variation.- 5. Signal-to-Noise Ratio.- 6. Matching Spectra to Available Light Sources.- 7. Stain Specificity.- 8. Diffusibility of Fluorophores.- 9. Autofluorescence.- D. DNA and RNA Determinations.- 1. DNA-Binding Dyes and Their Specificity of Binding.- 2. Analytical Considerations.- 3. Applications of DNA Staining in Single Cells.- a. Cell-Cycle Analysis.- b. DNA Synthesis.- c. Nuclear Ploidy and DNA Content of Isolated Nuclei.- 4. Multifunctional Staining (Single-Stranded and Double-Stranded Polynucleotide Fluorescence).- 5. Use of Two DNA-Binding Stains.- 6. Fluorescence Labeling of RNA.- E. Enzymes.- 1. Fluorogenic Substrates.- 2. Standardization of Enzyme Activity.- 3. Kinetic Measurements.- a. Substrate Diffusion into the Cell.- b. Enzyme-Substrate Reaction.- c. Product Diffusion.- 4. Multivariable Analysis.- 5. Cell Sorting to Validate Enzyme Analysis.- 6. Other Approaches to the Analysis of Enzymes in Single Cells.- F. Quantification of Proteins.- 1. Ionic Probes.- 2. Covalent Conjugates of Proteins and Fluorescent Ligands.- G. Conclusions.- References.- 7. Microspectrofluorometric Procedures and Their Applications in Biological Systems.- A. Introduction.- B. Methods.- 1. The Microspectrofluorometer.- a. Principle of the Instrument.- b. Microscopic Optical Arrangement.- i. Illuminator Blocks for Observations on Different Fluorochromes.- ii. Mirror and Dichromatic Options for Different Topographic Operations of the Instrument.- iii. Cell and Microinstrument Visualization.- c. Overall Construction Principle of the Optical Arrangement from Microscope to Detector. Conditions for Topographic and Spectral Operation.- d. Considerations Applied in Design.- i. The Overall Magnification and the Resolution of the Optical Multichannel Analyzer.- ii. Choice of Optical Components.- e. Signal-to-Noise Ratio.- f. Cell Tolerance to Exciting Wavelengths.- g. Conditions for Work with Exogenous Fluorochromes.- h. Electrical Design and Data Processing.- 2. Microinjection, Micromanipulatory Procedure.- 3. Biological Material. Cell Cultures and Observation Chambers.- 4. Morphological Studies of Microinjected Cells.- C. Results and Discussion.- 1. Kinetics of Intracellular, Transient Metabolic Processes.- 2. Rate-Controlling Factors.- 3. NADH-versus―NADPH Response.- 4. Compartmentalization and Intracellular Organelle Interactions.- 5. Multicellular Integrated States.- 6. Spectral Identification of Intracellular Coenzymes Found in Free States and Energy Transfer to Other Fluorochromes.- 7. Spectral Studies of Fluorescent Carcinogens.- a. Penetration.- b. Intracellular Distribution.- c. Intracellular Fate of Carcinogen Molecules.- D. Conclusion.- References.