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Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.