The QuanTI-FRET method proposes calibrating the instrument and the FRET pair to simply
calculate absolute FRET probabilities from triplet of images acquired under the same conditions
and with the same FRET pair. All the photophysical and instrumental factors are included in this calibration,
leaving
the variability of the results to biological origins.
The main contribution of the QuanTI-FRET method has been the single-step
calibration of the two correction factors based on the batching of millions of pixels, thus fully taking
advantage of the statistics of the imaged pixels and making this calibration method extremely robust.
The QuanTI-FRET has been reworked to allow a direct autocalibration from the
images of interest! It is now possible to avoid the preparation and imaging of FRET standards. We demonstrated
this idea with intramolecular FRET biosensors imaged in living cells with a relatively low fluorescence
signal. This autocalibration is possible because of the strong robustness of the QuanTIFRET calibration with
respect to the quality of the calibration dataset.
To make quantitative FRET measurement accessible to all laboratories with no coding expertise, we have
prepared a standalone in Python (GUI and CLI) and a plug-in for the Napari viewer. The code is open-source and made publicly
available, don't hesitate to contribute directly or through feedbacks.
"QuanTI-FRET: a framework for quantitative FRET measurements in living cells." A Coullomb, CM Bidan, C Qian, F Wehnekamp, C Oddou, C Albiges-Rizo, DC Lamb et A Dupont. Scientific Reports 10 (1), 1-11 (2020)
"Live-cell quantitative FRET imaging made simple by autocalibration in QuanTI-FRET." J Leblanc, AH Lombard, A Saumureau, S Costrel, J Revilloud, A Coullomb et A Dupont. The European Physical Journal E 48:74 (2025).
Co-authors of the code: Alexis Coullomb, Adrien Saumureau and Aurélie Dupont.