Spectrally resolved fluorescence lifetime imaging: New developments and applications

The fluorescence lifetime of different molecular species is calculated from the measured fluorescence intensity decrease following short pulsed laser excitation, by a multi-channel fitting procedure. In a FRET (Forster Resonant Energy Transfer) experiment the time dependent behaviour of the donor profile is assumed in a first view mono-exponential and the acceptor decay profile is solved analytically. A global minimization fitting algorithm has increased information content than a single channel fitting routine. In a normal FRET-FLIM experiment, the efficiency of FRET is calculated only by considering the kinetics of the donor. However, as will be shown, a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated.