Quantitative FRET data analysis: protein localization in living specimens

The current advances in fluorescence microscopy coupled with the development of new fluorescent probes and detectors provide the tools to study protein associations in living specimens using FRET microscopy. Upon energy transfer, donor fluorescence is quenched and acceptor fluorescence is increased (sensitized), resulting in a decrease in donor excitation intensity or lifetime. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal acquired using one- and two-photon excitation FRET microscopy. There are a number of methods to avoid, minimize or correct the spectral bleedthrough (SBT) contamination in intensity-based FRET, each having specific limitations depending on the level of sensitivity desired. We have developed an algorithm to correct the contamination in the FRET image to estimate the energy transfer efficiency (E%) and the distance (r) between donor and acceptor molecule. In this presentation we explain the influence of back-bleedthrough signal in quenched donor channel, acceptor excitation wavelength exciting donor component of the double labeled specimen (we call them additional SBT in this paper) and its influence in the calculation of energy transfer efficiency and the distance between donor and acceptor molecules. Considerable amount of additional SBT signals were observed in the intensity based multiphoton FRET microscopy compared to the one-photon FRET microscopy.