  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.
