Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence

Published

June 16, 2010

Author(s)

Katharine M. Mullen, Sergey Laptenok, Jan Willem , Antonie Visser, Ivo van Stokkum

Abstract

A methodology is described for the detection of F rster Resonance Energy Transfer (FRET) in live cells by monitoring the rise time of the acceptor fluorescence using fluorescence lifetime imaging microscopy (FLIM). An advantage of this method is that only those molecules are monitored that are involved in the energy-transfer process. The method relies on global analysis of FLIM images. As a result, a more accurate characterization of the amount of FRET occurring in a given system is obtained as compared to existing methodologies. Previous methods for the estimation of FRET efficiencies from FLIM data rely on estimation of the decay rate of donor fluorescence, which usually is a mixture of populations of donor molecules that are involved in FRET and those that are fluorescent but not involved in FRET. Alternatively, parameters describing the rise of acceptor fluorescence and the decay of the donor fluorescence can be determined via simultaneous global analysis of multiple FLIM images, thereby increasing the reliability of the analysis. In the present study, plant protoplasts transfected with fusions of visible fluorescent proteins are used to illustrate the new data analysis method. It is demonstrated that the distances estimated with the present method are substantially smaller than those estimated from the average donor lifetimes. Software to reproduce the presented results is provided in an open-source and freely available package called TIMP for The R project for Statistical Computing".

Citation

Physical Chemistry Chemical Physics

Volume

12

Pub Type

Journals

Keywords

FLIM, TIMP, ECFP, EYFP, EGFP, mCherry

Citation

Mullen, K. , Laptenok, S. , , J. , Visser, A. and van, I. (2010), Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence, Physical Chemistry Chemical Physics (Accessed November 8, 2024)