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
Table of Contents
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.
Department of Biochemistry, University of Oxford, Oxford, UK and St Anne's College, Oxford, UK.
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