QuanTI-FRET: a framework for quantitative FRET measurements in living cells

Abstract

Förster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instruments or limited by the complexity of the required analysis. With the recent development and expanding utilization of FRET-based biosensors, it becomes essential to allow biologists to produce quantitative results that can directly be compared. Here, we present a new calibration and analysis method allowing for quantitative FRET imaging in living cells with a simple fluorescence microscope. Aside from the spectral crosstalk corrections, two additional correction factors were defined from photophysical equations, describing the relative differences in excitation and detection efficiencies. The calibration is achieved in a single step, which renders the Quantitative Three-Image FRET (QuanTI-FRET) method extremely robust. The only requirement is a sample of known stoichiometry donor:acceptor, which is naturally the case for intramolecular FRET constructs. We show that QuanTI-FRET gives absolute FRET values, independent of the instrument or the expression level. Through the calculation of the stoichiometry, we assess the quality of the data thus making QuanTI-FRET usable confidently by non-specialists.

Simultaneous readout of multiple FRET pairs using photochromism

“…as is derived in Supplementary Note 1. γ can be estimated from model spectra, or more reliably from additional control experiments 10 .…”

“…as is derived in Supplementary Note 2. Equation (10) shows that the full dependence is known if one simply measures ρ 0 , the photoswitching ratio in the absence of FRET, using the same settings for the instrument. As we show in the Supplementary Information, this consideration can be expanded to photochromic molecules that do not display complete off-switching but instead reach an equilibrium or plateau level.…”

“…Most FRET experiments use a fluorescent donor and acceptor, which has the advantage that the FRET efficiency can be estimated based on the ratio between the donor-excited emission from both fluorophores [7][8][9][10] . The accuracy of this estimation depends on the ease with which the donor and acceptor can be spectrally separated from one another.…”

Simultaneous readout of multiple FRET pairs using photochromism

“…as is derived in section 1 of the supplementary information. can be estimated from model spectra, or more reliably from additional control experiments [10]. For some systems, such as genetically-encoded biosensors, changes in the observed FRET efficiency arise from the interconversion of the system between a low-and a high-FRET state, rather than from a continuous variation of the FRET efficiency.…”

“…as is derived in section 1 of the supplementary information. can be estimated from model spectra, or more reliably from additional control experiments [10].…”