Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells

European Biophysics Journal : EBJ
Andrew H A ClaytonEdouard C Nice

Abstract

Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.

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Citations

Mar 31, 2015·International Journal of Molecular Sciences·Dilip ShresthaJános Szöllősi
Jun 17, 2005·Journal of Microscopy·E B Van MunsterT W J Gadella
Dec 11, 2008·Microscopy Research and Technique·Michelle A Digman, Enrico Gratton
Aug 16, 2014·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Peter NagyJános Szöllősi
Jul 27, 2010·Biophysical Journal·Agnes SzabóPeter Nagy
Dec 22, 2007·Biophysical Journal·Michelle A DigmanEnrico Gratton
Sep 28, 2013·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Kai-Liang ChouJi-Yao Chen

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