Front Cover -- Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time Analysis -- Copyright Page -- Contents -- Series Preface -- Preface -- Contributors -- Part I: Introduction to Fluorescence Microscopy -- Chapter One. Fluorescence Microscopy -- 1.1 Introduction -- 1.2 Microscope design -- 1.3 Types of illumination -- 1.4 Light sources -- 1.5 Filters -- 1.6 Objectives and eyepieces -- References -- Part II: Optical Probes and Their Applications -- Chapter Two. Introduction to Fluorescent Probes: Properties, History and Applications -- 2.1 Introduction -- 2.2 Nature of fluorescence and properties of fluorescent probes -- 2.3 Historical developments -- 2.4 Applications of fluorochromes in histology and microbiology -- 2.5 Introduction of acridine orange into cell physiology, cytology and cytochemistry -- 2.6 General applications of fluorescent probes -- Acknowledgements -- References -- Chapter Three. Intracellular Ion Indicators -- 3.1 Introduction -- 3.2 General properties of intracellular ion indicators -- 3.3 Examples of intracellular ion indicators -- 3.4 Conclusions -- Acknowledgements -- References -- Chapter Four. Fluorescent Imaging of Nucleic Acids and Proteins in Gels -- 4.1 Introduction -- 4.2 General properties of fluorescent nucleic acid stains -- 4.3 Examples of fluorescent nucleic acid gel stains -- 4.4 General properties of fluorophore labels used to detect nucleic acids -- 4.5 General properties of fluorescent protein gel stains -- 4.6 Examples of fluorescent protein gel stains -- 4.7 Protein labelling -- 4.8 Conclusions -- Acknowledgements -- References -- Part III: Using Optical Probes in Cells - Practicalities, Problems and Pitfalls -- Chapter Five. Introducing and Calibrating Fluorescent Probes in Cells and Organelles -- 5.1 Introduction.

5.2 General principles of the loading process -- 5.3 General principles of the calibration process -- 5.4 Putting principles into practice -- Acknowledgements -- References -- Chapter Six. Electroporation: A Method for Introduction of Non-permeable Molecular Probes -- 6.1 Introduction -- 6.2 Basic concept of electroporation -- 6.3 Electric field generation and monitoring -- 6.4 Polarization of the outer membrane -- 6.5 Electropore formation and resealing -- 6.6 Transmembrane transport -- 6.7 Practical considerations of electroporation -- 6.8 Experimental evidence -- 6.9 Summary -- Acknowledgements -- References -- Chapter Seven. Imaging Reality: Understanding Maps of Physiological Cell Signals Measured by Fluorescence Microscopy and Digital Imaging -- 7.1 Introduction -- 7.2 Generic considerations for the use of fluorescent indicators -- 7.3 Optimization of fluorescent light detection and background light correction -- 7.4 3-D spatial maps of fluorescent signals -- Acknowledgements -- References -- Chapter Eight. Fluorescent Probes in Practice - Potential Artifacts -- 8.1 Introduction -- 8.2 Photobleaching -- 8.3 Dynamic range -- 8.4 Probe loading -- 8.5 Ion calibration -- 8.6 Cell movement and fast ion fluxes -- 8.7 Autofluorescence -- 8.8 Interactions between multiple probes -- 8.9 Averaging and intensifier noise -- 8.10 Probe leakage and exocytosis -- 8.11 Probe kinetics -- References -- Part IV. Optical Probes for Specific Molecules, Organelles and Cells -- Chapter Nine. Acridine Orange as a Probe for Cell and Molecular Biology -- 9.1 Introduction -- 9.2 Historical remarks -- 9.3 AO as a fluorescent dye -- 9.4 Spectral properties of AO in complexes with nucleic acids and other biopolymers -- 9.5 AO in the study of nucleic acids in vitro -- 9.6 AO in nucleic acid cytochemistry -- 9.7 AO DNA staining after acid pretreatments.

9.8 AO in the study of DNA thermal denaturation -- 9.9 AO in the study of the chromatin functional state -- 9.10 Fluorescence polarization of AO in studies of biopolymers -- 9.11 AO in chromosome banding -- 9.12 AO in acid polysaccharide histochemistry -- 9.13 AO binding to proteins -- 9.14 AO binding to a living cell -- 9.15 AO in the study of cell viability -- 9.16 AO in the study of apotopsis -- 9.17 AO in flow cytometry -- 9.18 Other applications of AO -- References -- Chapter Ten. Fluorescent Lipid Analogues: Applications in Cell and Membrane Biology -- 10.1 Introduction -- 10.2 Fluorescent lipid analogues -- 10.3 Applications -- Acknowledgements -- References -- Chapter Eleven. Optical Probes for Cyclic AMP -- 11.1 Rationale for creating optical probes for cyclic AMP -- 11.2 Previous methods for measuring cAMP or imaging related molecules -- 11.3 Alternative cAMP binding sites -- 11.4 Properties of A-kinase -- 11.5 Fluorescent labelling of A-kinase -- 11.6 Properties of FlCRhR -- 11.7 Introduction of FlCRhR into cells -- 11.8 Imaging of FlCRhR and free cAMP -- 11.9 Applications -- References -- Part V: Technology for Qualitative and Quantitative Detection of Optical Probes in Living Cells -- Chapter Twelve. Quantitative Digital Imaging of Biological Activity in Living Cells with Ion-sensitive Fluorescent Probes -- 12.1 Introduction -- 12.2 Fluorescent probes for living cell function -- 12.3 Observing biological activity in 'real time' -- 12.4 Ratiometric imaging of ion-sensitive flourescent probes -- 12.5 Imaging strategies -- 12.6 Digital image processing -- 12.7 Data presentation -- 12.8 Confocal laser scanning microscopy - optical approaches to enhanced image resolution -- 12.9 A novel high-speed digital confocal microscope -- 12.10 Digital deconvolution and digital confocal microscopy - 'soft' approaches to enhanced image resolution.

12.11 Fluorescent measurements of cytosolic ions - combined photometry with electrophysiology -- 12.12 Photometric measurements in single cells -- 12.13 Measurement of fluorescent light - photometry versus imaging -- 12.14 Photomultiplier tube technology -- 12.15 Photon counting versus photocurrent integration -- 12.16 Excitation filter switching -- 12.17 Dual-emission probes -- 12.18 Electrophysiology combined with photometry or imaging -- 12.19 Data acquisition -- 12.20 CONCORD - integrating imaging, photometry and electrophysiology on a single workstation for ion imaging experiments -- 12.21 Cell culture and loading of fluorescent probes -- 12.22 Calibration of ion-sensitive dyes in living cells and in solution -- 12.23 Deriving spectra data from optical probes in situ - the SpectralWIZARD -- 12.24 Summary -- References -- Chapter Thirteen. Fast Photometric Measurements of Cell Function Combined with Electrophysiology -- 13.1 Fluorescent light measurement -- 13.2 A fluorescence/electrophysiological recording system -- 13.3 The photomultiplier tube -- 13.4 Dual-emission dye measurement systems -- 13.5 Dual-excitation dye measurement systems -- 13.6 Analogue signal digitization -- 13.7 Fluorescence measurement systems -- 13.8 Software for recording fluorescence signals -- 13.9 The 'chart recorder' paradigm -- 13.10 The 'oscilloscope' paradigm -- 13.11 Leak current subtraction -- 13.12 Computer system designs -- 13.13 Conclusion -- References -- Equipment suppliers -- Chapter Fourteen. Potentiometric Membrane Dyes and Imaging Membrane Potential in Single Cells -- 14.1 Introduction -- 14.2 Optimization of dye indicator sensitivity -- 14.3 Mapping membrane potential by digital fluorescence microscopy -- 14.4 Conclusion -- Acknowledgements -- References -- Chapter Fifteen. Fast Multisite Optical Measurement of Membrane Potential, with Two Examples.

15.1 Introduction -- 15.2 Signal type -- 15.3 Dyes -- 15.4 Measuring technology -- 15.5 Two examples -- 15.6 Population signals from vertebrate brain -- 15.7 Future directions -- Acknowledgements -- References -- Chapter Sixteen. Imaging Membrane Potential Changes in Individual Neurons -- 16.1 Introduction -- 16.2 Extracellular application of voltage-sensitive dyes -- 16.3 Intracellular application of voltage-sensitive dyes -- 16.4 Dye injection -- 16.5 Optical recording -- 16.6 Multisite recording -- 16.7 Comparison of optical and electrical signals -- 16.8 Dye sensitivity (dF/F) and signal-to-noise ratio -- 16.9 Calibration of the voltage-sensitive dye measurements in terms of membrane potential -- 16.10 Pharmacological effects and photodynamic damage -- 16.11 Imaging spike trigger zone -- 16.12 Vertebrate neurons -- 16.13 Summary -- References -- Part VI: Using Novel Indications for Genetic, Molecular and Cellular Function -- Chapter Seventeen. Bioluminescent and Chemiluminescent Indicators for Molecular Signalling and Function in Living Cells -- 17.1 The natural history of bio- and chemiluminescence -- 17.2 The analytical potential of chemiluminescent compounds -- 17.3 Application of chemi- and bioluminescence to living cells -- 17.4 Bioluminescent reporter genes -- 17.5 Bioluminescent indicators for molecular signalling in live cells -- 17.6 Conclusions and future prospects -- References -- Chapter Eighteen. Luminescence Imaging of Gene Expression in Single Living Cells -- 18.1 Introduction -- 18.2 General considerations -- 18.3 Equipment required -- 18.4 Experimental procedures -- 18.5 Concluding remarks -- Acknowledgements -- References -- Chapter Nineteen. Enhanced Variants of the Green Fluorescent Protein for Greater Sensitivity, Different Colours and Detection of Apoptosis -- 19.1 Introduction -- 19.2 GFP variants.

19.3 Detection of apoptosis with GFP.