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Updated: Aug 9, 2025

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells
Published on: September 16, 2014
Reply to "Comment on 'Dependence of the Fluorescent Lifetime τ on the Concentration at High Dilution'": Extended
Heinz Langhals1, Thorben Schlücker1
1Department of Chemistry, LMU University of Munich, Butenandtstr. 13, Munich DE 81377, Germany.
Abstract:
In "Comment on 'Dependence of the Fluorescent Lifetime τ on the Concentration at High Dilution'" by Anne Myers Kelley and David F. Kelley, the newly found concentration dependence of the fluorescence lifetime was attributed to the reabsorption and the delay of re-emission of fluorescence light. Accordingly, a comparably high optical density is required for a damping of the optically exciting light beam, causing a special profile for the re-emitted light with partial multiple reabsorption. However, an extended recalculation and re-examination on the basis of experimental spectra and the initially published data indicated an only static filtering effect by some reabsorption of fluorescent light. The resulting dynamic refluorescence is isotropically emitted in all room directions where only a small share of 0.006-0.6% adds to the measured primary fluorescence so that an interference in the measurement of fluorescent lifetimes becomes unimportant. Thus, the initially published data were further supported. The discrepancy between the two controversial papers could be resolved in terms of the different considered optical densities: a comparably high optical density can explain the interpretation of Kelley and Kelley, whereas low optical densities enabled by the use of the highly fluorescent perylene dye support our interpretation of the concentration dependence of the fluorescent lifetime.
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