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.
