Fig. 1.

Schematic plot of FmFLIM system setup. Rotation of the polygon mirror (Lincoln Laser, spinning speed 10,000–55,000 rpm) introduces sweeping optical path difference, resulting in fast intensity modulation of the excitation lasers. As all laser lines experience the same amount of optical path difference at a given time, different lasers are modulated at distinct instantaneous frequencies. Fluorescence emission collected by PMTs is demodulated in reference to a specific excitation laser modulation. After mixing and filtering, only emission signal generated by the specific excitation laser remains. In the case of the acceptor within a FRET pair, fluorescent emission due to FRET process is obtained by mixing donor laser modulation with acceptor PMT signal; while emission from direct acceptor laser excitation is isolated by mixing acceptor laser modulation with acceptor PMT signal. BS = beam spliter, DM = dichroic mirror, F = filter, L = lens, M = mirror, PD = photodiode, PMT = photo multiplier tube.