Accurately measuring biochemical activity in living cells is a major challenge in bioimaging. FRET microscopy (Fluorescence Resonance Energy Transfer) is widely used for this purpose: it relies on energy transfer between two fluorophores in close proximity, making it possible to monitor molecular interactions or protein conformational changes in real time. While powerful, FRET remains difficult to exploit quantitatively, as results are often highly dependent on instrumental settings and experimental conditions.

To address these limitations, a multidisciplinary team of physicists and biologists from LIPHY, led by Aurélie Dupont, developed QuanTI-FRET, an innovative approach now available as a plug-in for napari.

The originality of the method lies in an auto-calibration strategy performed directly on the experimental images, exploiting the known stoichiometry of intramolecular FRET biosensors. This approach eliminates the need for additional calibration constructs and significantly simplifies the experimental workflow. Validated on live-cell FRET experiments, it enables the extraction of robust and comparable quantitative FRET values.

Released as open-source software and integrated into an open Python-based analysis environment, QuanTI-FRET contributes to making quantitative FRET imaging more accessible and reproducible for the bioimaging community.

Here’s the napari plug-in: https://napari-hub.org/plugins/quanti-fret.html

Interested to learn more? Read their scientific article here.