Description
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.
