As this method is non-invasive, it is most frequently used for live cell experiments. Because crosstalk is an important issue, controls and complex correction calculations are inherent to the method. Measurements are executed by detection of the fluorescent signals of the donor and FRET as well as acceptor in a line by line sequential scan acquisition.
Proper excitation conditions play a major role. In the first sequence, the donor must be excited to generate donor fluorescence and sensitized emission of the acceptor. In the second sequence, the selective excitation of the acceptor will create acceptor fluorescence.
FRET sample preparations must therefore include references of donor in the absence of the acceptor (donor only control) and acceptor in the absence of the donor (acceptor only control).
Ideally, all references are included in the same preparation. The donor and acceptor references are used to obtain calibration coefficients to correct for excitation and emission cross talk.
It is important to be aware that throughout the entire experiment and calibration routine, all measuring parameters such as gain, emission detection window, excitation intensities, zoom, format, scan speed, pinhole etc must remain constant.