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

TL;DR

QuanTI-FRET provides a simple, robust method for quantitative FRET imaging in living cells using standard microscopes, enabling accurate, instrument-independent measurements crucial for biosensor applications.

Contribution

It introduces a new calibration and analysis framework that allows for absolute, quantitative FRET measurements in living cells with minimal equipment and straightforward procedures.

Findings

Provides absolute FRET values independent of instrument variations.

Enables quantitative analysis with a single calibration step.

Applicable to non-specialists for live-cell biosensor studies.

Abstract

Foerster 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.