Schlachter S, Elder AD, Esposito A, Kaminski GS, Frank GH, van Geest LK, and Kaminski CF, "mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy", Opt Express, Volume 17, pp. 1557-1570, 2009, DOI:10.1364/OE.17.001557 |
Frequency-domain ﬂuorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring ﬂuorescence lifetimes in wideﬁeld microscopy. However, the resolution of multiple exponential ﬂuorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of ﬂuorophores without any a priori information. The system’s performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the ﬁrst practical application of an algorithm derived by G. Weber  for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.