Spectrophotometry and spectrofluorometry are core techniques used throughout biology and medicine. These techniques evolve continuously and this book provides information on the latest advances in spectroscopic methods. Light Spectroscopy begins by descr
Spectrophotometry and spectrofluorometry are core techniques used throughout biology and medicine. These techniques evolve continuously and this book provides information on the latest advances in spectroscopic methods. Light Spectroscopy begins by descrHinweis: Dieser Artikel kann nur an eine deutsche Lieferadresse ausgeliefert werden.
Department of Biochemistry, University of Oxford, Oxford, UK and St Anne's College, Oxford, UK.
Inhaltsangabe
Abbreviations Preface PART 1: BASIC PRINCIPLES AND METHODS 1. Principles of Spectrophotometry The nature of spectrophotometry Interaction of light with matter Absorption lines and absorption bands Absorption spectra Quantitative aspects of absorption Light emission in solution fluorescence Fluorescence spectra and the quantitation of fluorescence Light scattering revisited 2. What to Look at Chromophores Fluorophores Effect of environment on absorption Effect of environment on fluorescence Sensitivity and detection limits Intrinsic and extrinsic probes Indicator molecules 3. Spectrophotometer Design Components Light sources Wavelength selection The sample compartment Detectors Varying the wavelength (scanning) Data capture 4. Geometry, Light Paths and Beam Splitting Sources of error in absorbance measurements The single beam spectrophotometer The double beam spectrophotometer Variations on a theme The dual wavelength spectrophotometer Sources of error in fluorescence measurements The spectrofluorometer The dual wavelength fluorometer Measurements on opaque samples 5. Measuring Absorbance and Fluorescence Calibrating the instrument Choosing the cuvette Preparing the sample PART 2: TECHNIQUES AND APPLICATIONS 6. Measuring an Absorption Spectrum Introduction Calibrating the instrument Selecting a sample Choice of operating conditions Determining a baseline Interpreting spectra Working without oxygen How to improve resolution 7. Measuring a Fluorescence Emission Spectrum Types of fluorescence spectrum Calibrating the fluorometer Preparing the sample Selecting the wavelengths and slitwidth for scanning Selecting the speed of scanning Photo bleaching Determining a baseline Determination of "max and peak intensity Absolute spectra The effect of temperature 8. Measurement at a Fixed Wavelength Introduction Assay design Quantitation of nonabsorbing material Calibration Additional considerations for fluorescence assays Assays in turbid solutions Spectrophotometric measurements of rates 9. Probing the Environment Environmentally sensitive chromophores Measuring ligand binding to proteins Measurement of the rate of ligand binding to a protein Measuring the concentration of ions inside cell compartments Types of optical probe Measurement of intravesicular pH using a distribution probe Measurement of transmembrane potential Fluorescence energy transfer as a molecular ruler Probing the structure of a four way DNA junction Appendices Appendix A: Glossary Appendix B: Suppliers Appendix C: Further reading Index.
Abbreviations Preface PART 1: BASIC PRINCIPLES AND METHODS 1. Principles of Spectrophotometry The nature of spectrophotometry Interaction of light with matter Absorption lines and absorption bands Absorption spectra Quantitative aspects of absorption Light emission in solution fluorescence Fluorescence spectra and the quantitation of fluorescence Light scattering revisited 2. What to Look at Chromophores Fluorophores Effect of environment on absorption Effect of environment on fluorescence Sensitivity and detection limits Intrinsic and extrinsic probes Indicator molecules 3. Spectrophotometer Design Components Light sources Wavelength selection The sample compartment Detectors Varying the wavelength (scanning) Data capture 4. Geometry, Light Paths and Beam Splitting Sources of error in absorbance measurements The single beam spectrophotometer The double beam spectrophotometer Variations on a theme The dual wavelength spectrophotometer Sources of error in fluorescence measurements The spectrofluorometer The dual wavelength fluorometer Measurements on opaque samples 5. Measuring Absorbance and Fluorescence Calibrating the instrument Choosing the cuvette Preparing the sample PART 2: TECHNIQUES AND APPLICATIONS 6. Measuring an Absorption Spectrum Introduction Calibrating the instrument Selecting a sample Choice of operating conditions Determining a baseline Interpreting spectra Working without oxygen How to improve resolution 7. Measuring a Fluorescence Emission Spectrum Types of fluorescence spectrum Calibrating the fluorometer Preparing the sample Selecting the wavelengths and slitwidth for scanning Selecting the speed of scanning Photo bleaching Determining a baseline Determination of "max and peak intensity Absolute spectra The effect of temperature 8. Measurement at a Fixed Wavelength Introduction Assay design Quantitation of nonabsorbing material Calibration Additional considerations for fluorescence assays Assays in turbid solutions Spectrophotometric measurements of rates 9. Probing the Environment Environmentally sensitive chromophores Measuring ligand binding to proteins Measurement of the rate of ligand binding to a protein Measuring the concentration of ions inside cell compartments Types of optical probe Measurement of intravesicular pH using a distribution probe Measurement of transmembrane potential Fluorescence energy transfer as a molecular ruler Probing the structure of a four way DNA junction Appendices Appendix A: Glossary Appendix B: Suppliers Appendix C: Further reading Index.
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