Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells

Abstract

Fluorescent protein-based FRET is a powerful method for visualizing protein-protein interactions and biochemical reactions in living cells. It can be difficult, however, to avoid photobleaching when observing fluorescent cells under the microscope, especially those expressing CFP. We compared the sensitivity of two protein-based FRET pairs to light-induced fluorescence changes in the donor, on FRET determination by fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low excitation light levels of the time- and space-correlated single photon counting (TSCSPC) method, FLIM acquisitions were achieved without donor photobleaching. Here, we show that photobleaching of CFP by a mercury lamp under the microscope induced a decrease in the mean fluorescence lifetime, which interfered with FRET determination between CFP and YFP. Importantly, the range of light-induced variation of the mean fluorescence lifetime of CFP was not proportional to the decrease in the steady state fluorescence intensity and varied from cell to cell. The choice of the CFP/YFP pair therefore requires that the cells be observed and analyzed at very low light levels during the whole FRET experiment. In contrast, the GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if some GFP photobleaching took place. We thus demonstrate that CFP can be an unreliable donor for FRET determination in living cells, due to its photosensitivity properties. We demonstrate that the GFP/mCherry pair is better suited for FRET measurement by FLIM in living cells than the CFP/YFP pair.

High throughput FRET analysis of protein–protein interactions by slide‐based imaging laser scanning cytometry

“…E is a monotonously decreasing function of the a factor [Eq. (15)], which itself depends linearly on e 2 , the ratio of the extinction coefficients of ECFP and EYFP at the donor excitation wavelength, 458 nm [Eqs. (12) and (13) (47)), e 2 cannot be determined unequivocally.…”

“…In case only a fraction of donors is in complex with an acceptor, the gained FRET efficiency can be considered as a lower limit of the real E value. To resolve donor subpopulations characterized by different E values, fluorescence lifetime measurements (15)(16)(17) or single molecule conditions are needed (18,19).…”

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…Another frequently used combination is EGFP and the red fluorescent protein (RFP). Studies of proteins tagged with EGFP and monomeric RFP (mRFP) have already shown the potential use of this pair for FRET-FLIM applications (Peter et al 2005;Tramier et al 2006;Albertazzi et al 2009). …”

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

“…due to tautomerisation, protonation or ion binding) mean that the fluorescence decay is often complex. Such dynamic complexity must be taken into consideration when extracting quantitative information from FRET measurements, and this is an emerging area of research [12][13][14][15][16].…”