Abstract
FLIM-FRET is a powerful imaging technique to study molecular interactions and conformational changes in biological systems. However, current FLIM-FRET techniques have difficulties to provide robust quantitative analysis of heterogeneous protein species due to several factors arising from live cell imaging, including low signal to noise ratio, small lifetime differences and intensity imbalance between high and low FRET species. Here we present a FRET lifetime analysis method based on frequency domain Fourier lifetime excitation-emission matrix (FLEEM) imaging. FLEEM is a novel high-speed multiplexed frequency-domain lifetime imaging method developed by our group. With FLEEM, time-resolved fluorescence images of live cells can be acquired simultaneously in multiple excitation-emission channels, including the donor spontaneous emission, acceptor spontaneous emission induced by direct acceptor excitation and the sensitized emission from the acceptor induced by FRET1. Assuming a two conformation model, each protein conformation has a distinct FRET efficiency, which results in two distinct donor lifetimes. The measured average donor lifetimes in both donor spontaneous emission and FRET-induced sensitized emission channel are population weighted averages of these two lifetimes, readily described by a two-dimension linear parametric function, of which weighting coefficients of two lifetime species differ between two channels. Two FRET efficiencies corresponding to low and high FRET conformation can be extracted from parametric fitting of the experimentally measured life times. The ratio of the two conformation populations can then be easily computed from the average donor lifetime. We used this method to analyze time-resolved images of FRET sensors in live HeLa cells and obtained quantitative measurements of sensor FRET efficiencies under reacted or unreacted states, which allow time-lapse quantifications of Ca2+ and cAMP levels in vivo.1. Zhao, M., Huang, R. & Peng, L. Opt. Express20, 26806-27 (2012).
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