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Abstract
Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mfD. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels Nch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, Nch or small fractions of interacting donor fD). The validity domain of the moments method is more restricted (not applicable when fD<0.25 or when Nch = 4) but it is more precise than the polar approach. We also demonstrate that the mfD is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.
Highlights
Forster Resonance Energy Transfer (FRET) is a photo-physical phenomenon in which energy is non-radiatively transferred from one excited fluorescent donor molecule to a nearby acceptor
We applied these non-fitting strategies to quantify Rac-GTPase activity by using a PBD assay [24] and we have successfully evaluated the spatiotemporal activation of Rac by measuring the FRET signal with our fast-fluorescence lifetime imaging microscopy (FLIM) prototype capable of acquiring FLIM images at high speed (,1 FLIM image/sec)
The real fraction of interacting donor (fD) contribution for each mixture has been calculated by taking into account the pre-exponential factor coming from each pure fluorescence decay (a = I/t where I corresponds to the total integral intensity and t is the fluorescence lifetime), we found fD = 0.17, 0.44 and 0.65
Summary
Forster Resonance Energy Transfer (FRET) is a photo-physical phenomenon in which energy is non-radiatively transferred from one excited fluorescent donor molecule to a nearby acceptor It depends on both the distance and the relative orientation of the two fluorophores (donor and acceptor) and occurs efficiently when the distance between the two molecules is less than approximately 10 nm [1], a distance comparable to the dimensions of biological macromolecules. We investigate FRET by measuring the donor fluorescence lifetime with fluorescence lifetime imaging microscopy (FLIM) This technique is advantageous for measuring FRET compared to the intensity based approaches since the lifetime is a spectroscopic property which is insensitive to the donor concentration, the donor-acceptor stoichiometry and the excitation intensity fluctuations
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