Modern Fluorescence Spectroscopy

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.