Light Spectroscopy

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

• 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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