1 Historical Aspects of Fluorescence.- 1 Introduction: On the Origin of the Terms Fluorescence, Phosphorescence, and Luminescence.- References.- 2 Pioneering Contributions of Jean and Francis Perrin to Molecular Luminescence.- 2.1 Introduction.- 2.2 Biographical Sketches of Jean Perrin and Francis Perrin.- 2.3 The Perrin-Jablonski Diagram.- 2.3.1 Jablonski Diagram.- 2.3.2 États Métastables — Phosphorescence.- 2.4 Resonance Energy Transfer.- 2.5 Fluorescence Polarization.- 2.6 Concluding Remarks.- 2.7 Bibliographical Notes.- References.- 3 The Seminal Contributions of Gregorio Weber to Modern Fluorescence Spectroscopy.- 3.1 Overview.- 3.2 EarlyYears.- 3.3 Cambridge.- 3.4 Francis Perrin’s Influence.- 3.5 Ph.D. Thesis.- 3.6 Postdoctoral.- 3.7 Sheffield.- 3.8 Intrinsic Protein Fluorescence.- 3.9 Red-Edge Effects.- 3.10 EEM.- 3.11 Brandeis.- 3.12 University of Illinois.- 3.13 Phase Fluorometry.- 3.14 Polarization Revisited.- 3.15 Students, Postdocs and Visitors.- 3.16 Commercialization of Fluorescence.- 3.17 National Laboratories.- 3.18 Honors.- 3.19 Proteins and Pressure.- References.- 2 Fluorescence of Molecular and Supramolecular Systems.- 4 Investigation of Femtosecond Chemical Reactivity by Means of Fluorescence Up-Conversion.- 4.1 Nanosecond and Picosecond Time-Resolved Fluorescence Techniques.- 4.1.1 Phase Modulation Spectroscopy.- 4.1.2 Time Correlated Single Photon Counting.- 4.1.3 Streak Cameras for Time-Domain Measurements.- 4.2 Femtosecond Emission Spectroscopy by Time-Gated Up-Conversion.- 4.2.1 Historical Background of the Time-Gated Up-Conversion Technique.- 4.2.2 Principle of the Time-Gated Up-Conversion Technique.- 4.2.2.1 Phase Matching Conditions.- 4.2.2.2 Quantum Efficiency for Up-Conversion.- 4.2.2.3 Group Velocity Effects.- 4.2.3 Experimental Setup.- 4.3 Time-Resolved Spectroscopy.- 4.3.1 Solvation Processes.- 4.3.1.1 Time-Dependent Fluorescence Stokes Shift (TDFSS) Non-Specific Solvation.- 4.3.1.2 Specific Solvation: Role of the Structure and the Charge of the Probe.- 4.3.1.3 Specific Solvation: Hydrogen Bond Dynamics.- 4.3.1.4 Isotope Effect.- 4.3.1.5 Spectral Narrowing in the 10 ps Time Scale.- 4.3.2 Photoinduced Intramolecular Charge Transfer.- 4.3.3 Intermolecular Electron Transfer.- 4.3.4 Intramolecular Proton Transfer.- 4.3.5 S2?S1 Internal Conversion.- 4.3.6 Biological Systems.- 4.4 Conclusions.- References.- 5 Spectroscopic Investigations of Intermolecular Interactions in Supercritical Fluids.- 5.1 Introduction.- 5.2 Instrumentation.- 5.3 Sample Preparation and Precautions..- 5.4 Selected Applications.- 5.5 Laser Flash Photolysis.- 5.6 Basic Picture Revealed by These Studies.- 5.7 The Future.- References.- 6 Space and Time Resolved Spectroscopy of Two-Dimensional Molecular Assemblies.- 6.1 Introduction.- 6.1.1 Motivation.- 6.1.2 Models.- 6.2 Experimental.- 6.3 Results and Discussion.- 6.3.1 Inhomogeneous Multilayers: RB 18 and ARA.- 6.3.2 Homogeneous Multilayers: SRH+ARA.- 6.3.3 Multilayers of CV18 and ARA or DPPA.- 6.3.3.1 CV 18 in DPPA.- 6.3.3.2 Cd-Arachidate Multilayers.- 6.3.4 Intralayer Quenching of PYR18 by CV18.- 6.4 Conclusions.- References.- 7 From Cyanines to Styryl Bases — Photophysical Properties, Photochemical Mechanisms, and Cation Sensing Abilities of Charged and Neutral Polymethinic Dyes.- 7.1 Introduction.- 7.2 Cyanine Dyes.- 7.2.1 Photophysical Model Mechanisms.- 7.2.2 Complexation Properties.- 7.3 Styryl Dyes.- 7.3.1 Photophysical Model Mechanisms.- 7.3.2 Complexation Properties.- 7.4 Styryl Bases.- 7.4.1 Photophysical Model Mechanisms.- 7.4.2 Complexation Properties.- 7.4.2.1 Donor Acceptor Fluoroionophores.- 7.4.2.2 Donor Acceptor Donor Fluoroionophores.- 7.5 Conclusion.- References.- 8 Phototunable Metal Cation Binding Ability of Some Fluorescent Macrocydic Ditopic Receptors.- 8.1 Introduction.- 8.2 Anthraceno Coronands.- 8.2.1 Free Ligand.- 8.2.2 In the Presence of Metal Cation.- 8.3 Benzeno Coronands.- 8.3.1 BBO5O5.- 8.3.2 0TTO5O5.- 8.3.3 Fluorescence Anisotropy Experiments with BBO5O5.- 8.4 Conclusion.- References.- 3 Fluorescence in Sensing Applications.- 9 The Design of Molecular Artificial Sugar Sensing Systems.- 9.1 Introduction.- 9.2 Fluorescent Monoboronic Acids.- 9.3 Selective Recognition of Saccharides by Diboronic Acids.- 9.4 Introduction of the Concept of PET (Photoinduced Electron Transfer) Sensors.- 9.5 A Glucose Sensor and an Enantioselective Sensor.- 9.6 Conclusion.- References.- 10 PCT (Photoinduced Charge Transfer) Fluorescent Molecular Sensors for Cation Recognition.- 10.1 Introduction.- 10.2 Principles.- 10.3 PCT Sensors Based on the Interaction Between the Bound Cation and an Electron-Donating Group.- 10.3.1 Crown-Containing PCT Sensors.- 10.3.2 Chelating PCT Sensors.- 10.3.3 Cryptand-Based PCT Sensors.- 10.3.4 Calixarene-Based PCT Sensors.- 10.4 PCT Sensors Based on the Interaction Between the Bound Cation and an Electron-Withdrawing Group.- 10.4.1 Crown-Containing PCT Sensors.- 10.4.2 Calixarene-Based PCT Sensors.- 10.5 Conclusion.- References.- 11 Fluorometric Detection of Anion Activity and Temperature Changes.- 11.1 The Two-Component Approach to the Design of a Fluorescent Molecular Sensor.- 11.2 The Use of a [ZnII(tren)]2+ Platform for Anion Recognition and Fluorescent Sensing.- 11.3 Carboxylate Recognition Signalled by Fluorescence Enhancement.- 11.4 The Design of a Molecular Fluorescent Thermometer.- References.- 12 Oxygen Diffusion in Polymer Films for Luminescence Barometry Applications.- 12.1 Introduction.- 12.1.1 Measuring Oxygen Transport.- 12.2 Oxygen Diffusion and Luminescence Quenching.- 12.2.1 Diffusion-Controlled Reactions.- 12.2.2 Quenching and Oxygen Diffusion.- 12.3 Silicone Polymers.- 12.3.1 PDMS.- 12.3.2 Genesee Resins.- 12.4 Poly(aminothionylphosphazenes) (PATP).- 12.5 Modified Poly(aminothionylphosphazenes).- 12.5.1 MSPTP.- 12.5.2 PTHF.- 12.5.3 C4PATP-PTHF Block Copolymers.- 12.5.4 MSPTP-PTHF.- 12.6 Summary.- References.- 13 Dual Lifetime Referencing (DLR) — a New Scheme for Converting Fluorescence Intensity into a Frequency- Domain or Time-Domain Information.- 13.1 Introduction.- 13.2 Theoretical Background.- 13.2.1 Frequency Domain DLR Spectroscopy.- 13.2.2 Time-Domain DLR Spectroscopy.- 13.3 Phosphorescent Standards.- 13.4 Instrumentation.- 13.5 DLR Applications.- 13.5.1 Homogeneous Assays.- 13.5.2 DLR Based Optical Sensors.- 13.5.2.1 Optical Chloride Sensor Based on DLR.- 13.5.2.2 Fiber Optic pCO2 Microsensor Based on DLR.- 13.5.3 DLR Imaging Using Planar Optical pH Sensors.- 13.5.4 Outlook.- References.- 4 New Techniques of Fluorescence Microscopy in Biology.- 14 Two-Photon Fluorescence Fluctuation Spectroscopy.- 14.1 Introduction.- 14.2 Instrumentation.- 14.2.1 Laser.- 14.2.2 Microscope Objectives.- 14.2.3 Microscope, Filters, and Electronics.- 14.3 Autocorrelation.- 14.3.1 Single Species.- 14.3.2 Calibration of the Excitation Volume.- 14.3.3 Comparison of Models.- 14.3.4 Multiple Species.- 14.4 Moment Analysis.- 14.4.1 Comparison Between PCH and Moment Analysis.- 14.5 Conclusions.- References.- 15 Fluorescence Lifetime Imaging Microscopy of Signal Transduction Protein Reactions in Cells.- 15.1 Imaging Protein States by FRET.- 15.2 FRET Imaging by Donor Fluorescence Lifetime.- 15.3 Acceptor Photobleaching in FRET Imaging.- 15.4 Fluorescence Lifetime Imaging Microscopy.- 15.5 Global Analysis and the Population of States.- 15.6 Conclusions.- References.- 16 New Techniques for DNA Sequencing Based on Diode Laser Excitation and Time-Resolved Fluorescence Detection.- 16.1 Introduction.- 16.1.1 The Multiplex Dye Principle and Pattern Recognition.- 16.2 DNA Sequencing in Capillary Gel Electrophoresis by Diode Laser-Based Time-Resolved Fluorescence Detection.- 16.2.1 Semiconductor Lasers as Efficient Excitation Source in the Red Spectral Region.- 16.2.2 Design of Multiplex DNA Sequencing Primers.- 16.2.3 4-Dye-1-Lane Multiplex DNA Sequencing.- 16.3 High-Throughput DNA Analysis.- 16.3.1 Increasing the Speed of Electrophoresis.- 16.3.2 Construction of an Ideal Capillary Array Electrophoresis Instrument (CAE).- 16.3.3 Capillary Array Scanner for Time—Resolved Fluorescence Detection.- 16.3.3.1 Discontinuous Bidirectional Scanning.- 16.3.3.2 Time-Resolved Detection in Parallel Capillaries.- 16.4 Sequencing by Hybridization (SBH).- 16.5 Single Molecule DNA Sequencing in Submicrometer Channels.- References.- 17 The Integration of Single Molecule Detection Technologies into Miniaturized Drug Screening: Current Status and Future Perspectives.- 17.1 Introduction.- 17.2 Theoretical Background of Common Approaches in Single Molecule Analysis (SMA).- 17.2.1 Principles of Fluorescence Correlation Spectroscopy (FCS).- 17.2.2 Autocorrelation Analysis.- 17.2.3 Features and Issues of FCS—Based Screening.- 17.2.4 Photon Counting Statistics: Poisson and Super-Poisson Analysis.- 17.2.5 Photon Counting Histogram (PCH).- 17.2.6 Fluorescence Intensity Distribution Analysis (FIDA).- 17.2.7 Features and Issues of FIDA and PCH.- 17.2.8 Burst Integrated Lifetime (BIFL).- 17.2.9 Features and Issues of BIFL.- 17.3 Conclusion and Outlook.- References.- 18 Picosecond Fluorescence Lifetime Imaging Spectroscopy as a New Tool for 3D Structure Determination of Macromolecules in Living Cells.- 18.1 Time- and Space-Correlated Single Photon Counting (TSCSPC) Spectroscopy and Microscopy.- 18.1.1 DL-System.- 18.1.2 QA-System.- 18.2 EC Biotechnology Demonstration Project: Picosecond Fluorescence Lifetime Imaging as a New Tool for 3 D Structure Determination of Macromolecules in Cells.- 18.2.1 Current State of Knowledge.- 18.2.2 Demonstration Objectives.- 18.2.3 Work Content.- 18.2.4 Role of Partners.- 18.2.4.1 Technology Producers.- 18.2.4.2 Technology Users.- 18.3 Multi-Parameter TSCSPC.- 18.4 Minimal-Invasive Fluorescence Microscopy (MIFM).- 18.5 Living Cells: Fluorescence Dynamics Imaging.- 18.5.1 Fluorescence and Fluorescence Anisotropy Decays of EB-Intercalated DNA in the Cell Nucleus: Collaboration with Maïté Coppey-Moisan (Institut Jacques Monod, Paris).- 18.5.2 GFP-Aggregation, Studied by Fluorescence and Fluorescence Anisotropy Dynamics: Collaboration with Maïté Coppey-Moisan (Institut Jacques Monod, Paris).- 18.5.3 Protein-Protein Interaction: Collaboration with Jürgen Bereiter-Hahn (Goethe University Frankfurt).- 18.5.4 Mitochondria: Fluorescence Dynamics of DASPMI and Rhodamine 700: Collaboration with Jürgen Bereiter-Hahn (Goethe University Frankfurt).- 18.5.5 Chloroplasts: Photosynthesis in Living Plant Cells by Observing Fluorescence Dynamics of the Reaction Centre in Individual Chloroplasts: Collaboration with Hann-Jörg Eckert (TU Berlin).- 18.5.6 The Acquisition of Fluorescence Lifetime Values from Intracellular Sulphonated Aluminium Phthalocyanines Using Confocal Point-Scan and Wide-Field QA Detection [32b] Collaboration with David Phillips (Imperial College, London).- 18.5.6.1 Application of the QA Detector to Obtaining Fluorescence Lifetime Values from Intracellular Sulphonated Aluminium Phthalocyanines.- 18.5.6.2 The Application of Confocal Fluorescence Microscopy in Obtaining Fluorescence Lifetime Values from Intracellular Sulphonated Aluminium Phthalocyanines.- 18.5.6.3 Interpretation of the Results in Terms of Intracellular Phthalocyanine Localisation.- 18.6 Vehicle Micro-Spectroscopy.- References.- 5 Proteins and Their Interactions as Studied by Fluorescence Methods.- 19 About the Prediction of Tryptophan Fluorescence Lifetimes and the Analysis of Fluorescence Changes in Multi-Tryptophan Proteins.- 19.1 Interpreting Fluorescence Changes in Proteins.- 19.2 Determination of the Parameters.- 19.2.1 The Wavelength-Independent Amplitude Fraction ?.- 19.2.2 The Radiative Rate Constant.- 19.3 Analysis of the Meaning of the Different Factors of Q/Q0.- 19.3.1 Heterogeneous Static Quenching or Population Reshuffling (fPR).- 19.3.1.1 Estimation of Microstates of Tryptophan Side Chains.- 19.3.2 The Factor of Pure Dynamic Quenching (fDQ).- 19.4 Examples.- 19.5 Comparison of a System with multiple Fluorophores and Multiple Lifetimes with a System Containing One Fluorophore with Multiple Lifetimes.- 19.5.1 Examples.- 19.6 Conclusion.- References.- 20 Application of Time-Resolved Fluorescence Spectroscopy to Studies of DNA-Protein Interactions and RNA Folding.- 20.1 Introduction.- 20.2 DNA Polymerase Proofreading.- 20.2.1 Detecting the Two DNA Binding Modes of Klenow Fragment.- 20.2.2 Time-Resolved Anisotropy for a Heterogeneous Mixture of Probe Environments.- 20.2.3 Partitioning of Mismatched DNA Substrates Between pol and exo Sites.- 20.2.4 Energetic Contributions of Protein Side Chains to DNA Partitioning.- 20.3 Tertiary Structure Formation in the Hairpin Ribozyme.- 20.3.1 tr-FRET Analysis of the Hairpin Ribozyme.- 20.3.2 Influence of the Interdomain Junction on Ribozyme Folding.- 20.4 Conclusions and Outlook.- References.- 21 Rare Earth Cryptates and TRACE Technology as Tools for Probing Molecular Interactions in Biology.- 21.1 Introduction.- 21.2 Fluorescence and Homogeneous Assays.- 21.2.1 Time Resolved Fluorescence and Rare Earth Complexes.- 21.2.2 Rare Earth Chelates.- 21.3 TRACE Technology.- 21.3.1 Rare Earth Cryptates as a New Type of Fluorescent Label.- 21.3.2 Modulation Processes and Homogeneous Assays.- 21.3.3 Dual Wavelength Detection.- 21.3.4 TRACE Application in Immunoanalysis.- 21.3.5 Kinetic Measurements.- 21.4 TRACE for Probing Molecular Interactions in Life Science.- 21.4.1 Cell Surface Receptor Studies.- 21.4.2 Receptor Tyrosine Kinase Assay.- 21.4.3 Protein-Protein Interactions.- 21.4.4 Protease Assays.- 21.4.5 Applications in Molecular Biochemistry.- 21.4.5.1 Nucleic Acid Hybridization.- 21.4.5.2 Incorporation of TBP Eu3+ Labeled Nucleotides in DNA and RNA.- 21.4.6 “Cassettes” Formats as a Generic Tool.- 21.5 Conclusion.- References.- 22 Tracking Molecular Dynamics of Flavoproteins with Time-Resolved Fluorescence Spectroscopy.- 22.1 Intrinsic Protein Fluorescence.- 22.2 Flavins and Flavoproteins.- 22.3 Flavin as a Fluorescent Probe for Flavoprotein Dynamics.- 22.4 The Intrinsic Flexibility of Proteins.- 22.5 Functionally Important Motions in Flavoenzymes; an Introduction to Glutathione Reductase, Thioredoxin Reductase and p-Hydroxybenzoate Hydroxylase.- 22.6 Current Insights in Flavoprotein Active-Site Dynamics from Fluorescence: the Drive to Higher Time-Resolution, the Revised Interpretation of Heterogeneous Fluorescence Decays, and the Introduction of a New Mechanism for Fluorescence Depolarization.- 22.7 From Ensembles to Single Molecules.- 22.8 Prospects for Studying Conformational Dynamics by Flavin Fluorescence Detection.- References.