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QuanTI-FRET, a new method for the quantitative analysis of FRET in living cells

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FRET, Förster Resonance Energy Transfer, is an energy transfer between two fluorophores that only takes place when they are within 10nm of each other. The quantitative measurement of this effect makes it possible to measure distances in the nanometer range with a simple fluorescence microscope. While this was possible for isolated molecules, quantification of FRET was considered almost impossible in more complex samples. A team at LIPhy has developed a new method for quantitative measurements of FRET in living cells. This paves the way for a more extensive and successful use of fluorescent biosensors.

In single molecule experiments, it is possible to measure distances of a few nanometers with sub-nanometer accuracy in solution on dynamic molecules. This achievement is possible thanks to the FRET phenomenon and thanks to a special methodology for the acquisition of photons and the analysis of their origin. The interest for FRET to probe living organisms has grown in recent years with the development of genetically encoded fluorescent biosensors. However, the measurement of these probes remained qualitative at best, if not biased, thus slowing down the development of the very promising FRET biosensors.
In partnership with a team from the IAB and a team from the LMU (Munich), a team from LIPhy has taken the methods used in single molecule to adapt them to the microscopy of living cells. The QuanTI-FRET method makes it possible to calibrate an experimental system with correction factors that are directly measured but, derived from photophysical equations describing the different excitation and emission efficiencies. Beyond the calibration of spectral crosstalks, the heart of the calculation is carried out in a single step making the QuanTI-FRET method robust. Thus, with an imaging protocol in three different spectral channels, we obtain a quantitative FRET efficiency map even in living cells (Fig.1). This has been demonstrated by carrying out two completely independent measurement campaigns, one in Grenoble and the other in Munich, and by obtaining the same absolute FRET values for FRET standards.
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. The QuanTI-FRET method provides absolute FRET values, independent of instrument and expression level. By calculating the relative stoichiometry of fluorophores, an estimate of the quality of the data is provided, making QuanTIFRET usable confidently by non-specialists.

Measurements of FRET standards for three distances (C5V, C17V et C32V) . The fluorescence images were taken in three spectral channels and analyzed with QuanTI-FRET resulting in a quantitative FRET map. Scale bar 20µm.

View online : QuanTi-FRET: a framework for quantitative fRet measurements in living cells, A. Coullomb, C. M. Bidan, C. Qian, F. Wehnekamp, C. Oddou, C. Albigès-Rizo, D.Lamb & A. Dupont, Sci. Rep. April 2020