Chapter 6. Principles of Steady-State and Time-Resolved Fluorometric Techniques

  1. Prof. Dr. Bernard Valeur

Published Online: 20 DEC 2001

DOI: 10.1002/3527600248.ch6

Molecular Fluorescence: Principles and Applications

Molecular Fluorescence: Principles and Applications

How to Cite

Valeur, B. (2001) Principles of Steady-State and Time-Resolved Fluorometric Techniques, in Molecular Fluorescence: Principles and Applications, Wiley-VCH Verlag GmbH, Weinheim, FRG. doi: 10.1002/3527600248.ch6

Author Information

  1. Laboratoire de Chimie Générale, Conservatoire National des Arts et Métiers, 292 rue Saint-Martin, 75141 Paris Cedex 03, France

Publication History

  1. Published Online: 20 DEC 2001
  2. Published Print: 21 DEC 2001

ISBN Information

Print ISBN: 9783527299195

Online ISBN: 9783527600243

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Keywords:

  • steady-state spectrofluorometry;
  • spectrofluorometer;
  • excitation spectra;
  • emission spectra;
  • inner filter effects;
  • polarization effects;
  • polarization spectra;
  • time-resolved fluorometry;
  • phase-modulation fluorometers;
  • pulse fluorometers;
  • electro-optic modulator;
  • complex fluorescence decays;
  • lifetime distributions

Summary

  • Steady-state spectrofluorometry

    • Operating principles of a spectrofluorometer

    • Correction of excitation spectra

    • Correction of emission spectra

    • Measurement of fluorescence quantum yields

    • Problems in steady-state fluorescence measurements: inner filter effects and polarization effects

    • Measurement of steady-state emission anisotropy. Polarization spectra

  • Time-resolved fluorometry

    • General principles of pulse and phase-modulation fluorometries

    • Design of pulse fluorometers

      • Single-photon timing technique

      • Stroboscopic technique

      • Other techniques

    • Design of phase-modulation fluorometers

      • Phase fluorometers using a continuous light source and an electro-optic modulator

      • Phase fluorometers using the harmonic content of a pulsed laser

    • Problems with data collection by pulse and phase-modulation fluorometers

      • Dependence of the instrument response on wavelength. Color effect

      • Polarization effects

      • Effect of light scattering

    • Data analysis

      • Pulse fluorometry

      • Phase-modulation fluorometry

      • Judging the quality of the fit

      • Global analysis

      • Complex fluorescence decays. Lifetime distributions

    • Lifetime standards

    • Time-dependent anisotropy measurements

      • Pulse fluorometry

      • Phase-modulation fluorometry

    • Time-resolved fluorescence spectra

    • Lifetime-based decomposition of spectra

  • Comparison between pulse and phase fluorometries

  • Appendix: Elimination of polarization effects in the measurement of fluorescence intensity and lifetime