Using FLIM, the FRET efficiency is calculated from the difference of donor lifetime in absence and presence of the acceptor. For reliable quantification the detection window must match the donor emission spectrum. It must exclude any other fluorescence, such as autofluorescence or acceptor fluorescence.
Finding the correct detection range can be time-consuming when band pass filters have to be switched. A faster and more convenient approach is based on spectrally resolved detection. The FLIM wizard contains a xyλ-mode which allows the user to automate recording of a wavelength scan. This option is exclusively available to systems containing internal SP FLIM detectors.
Sample: Identify optimal spectral detection range for unbiased FRET (see steps 1 to 4 ).
GFP, GFP-mCherry tandem, GFP+mCherry were expressed in HeLa cells. A FLIM λ series of the three samples was acquired. The plot of average lifetime over detection wavelength shows that the optimal FRET detection window ranges from 485–545 nm (constant values). Courtesy: M. Weiss, J. Szymanski, DKFZ, Heidelberg.