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Updated: Aug 17, 2026

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
Published on: February 9, 2012
Pitfalls and their remedies in time-resolved fluorescence spectroscopy and microscopy
Martin vandeVen1, Marcel Ameloot, Bernard Valeur
1Biomedisch Onderzoeksinstituut, Limburgs Universitair Centrum, School of Life Sciences, Transnationale Universiteit Limburg, 3590 Diepenbeek, Belgium.
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.
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