Journal of Fluorescence

, Volume 15, Issue 3, pp 377–413 | Cite as

Pitfalls and Their Remedies in Time-Resolved Fluorescence Spectroscopy and Microscopy

  • Martin vandeVen
  • Marcel Ameloot
  • Bernard Valeur
  • Noël Boens
Article

Abstract

Time-resolved fluorescence spectroscopy and microscopy in both time and frequency domains provide very useful and accurate information on dynamic processes. Good quality data are essential in obtaining reliable parameter estimates. Distortions of the fluorescence response due to artifacts may have disastrous consequences. We provide here a concise overview of potential difficulties encountered under daily laboratory circumstances in the use of time- and frequency-domain equipment as well as practical remedies against common error conditions, elucidated with several graphs to aid the researcher in visual inspection and quality-control of collected data. A range of artifacts due to sample preparation or to optical and electronic pitfalls are discussed, as are remedies against them. Also recommended data analysis strategies are described.

Key Words

Time-resolved fluorescence spectroscopy time-resolved fluorescence microscopy artifacts pulse fluorometry phase-modulation fluorometry FLIM single-photon timing sample preparation optics electronics data analysis 

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Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Martin vandeVen
    • 1
  • Marcel Ameloot
    • 1
  • Bernard Valeur
    • 2
    • 3
  • Noël Boens
    • 4
  1. 1.Biomedisch Onderzoeksinstituut, Limburgs Universitair Centrum, School of Life SciencesTransnationale Universiteit LimburgDiepenbeekBelgium
  2. 2.CNRS UMR 8531, Laboratoire de Chimie généraleCNAMParis cedex 03France
  3. 3.CNRS UMR 8531, Laboratoire PPSMENS-CachanCachan cedexFrance
  4. 4.Department of ChemistryKatholieke Universiteit LeuvenHeverleeBelgium

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