827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

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.

Fast bi-exponential fluorescence lifetime imaging analysis methods

“…4(g), consistent with the typical lifetime of two-photon luminescence of GNRs as reported previously [4]. The bias can be corrected using the method introduced in [12] or a look-up table for faster corrections [7]. Figures 4(b), 4(i), and 4(j) show that Phasor is biased when τ F ≪ τ D making it difficult to locate GNRs precisely.…”

“…These hardware friendly algorithms mainly serve to (1) compress the raw data to enhance the frame rate and to release the need for high-speed serial links between the camera and a workstation and (2) generate fast FLIM imaging with the minimum latency between the data collection and image generation for real-time applications. In this Letter we will propose new hardware friendly algorithms and compare their performances with some recently published nonfitting algorithms [11][12][13].…”

“…lifetimes, K is the pre-scalar, f D the proportion, ut the step function, and we neglect the instrumental response function (IRF) and background noise as the model described in [12]. The assumption allows a proper comparison with other algorithms and provides intuitive insights for choosing a proper strategy in system design and data acquisition.…”

“…Similar to the phasor approach (Phasor) and moment method (MoM) [12,13], we can fix τ D to obtain τ F and f D as Eqs. (5) and (6),…”

“…MoM is the most biased among all methods, regardless of M, and worse than BCMM 2 although they both use the second moment. The integral of the second moment, Y 0 ,i nT;∞ used in MoM cannot be ignored and needs to be corrected [12]. Simpson's rule is useful when a gated or TCSPC system only has a limited number of temporal channels.…”