mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy.
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 40 MHz 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 [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.
Schlachter
S
S
Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK. ss678@cam.ac.uk
Elder
A D
AD
Esposito
A
A
Kaminski
G S
GS
Frank
J H
JH
van Geest
L K
LK
Kaminski
C F
CF
D000465:Algorithms; D002138:Calibration; D045744:Cell Line, Tumor; D004396:Coloring Agents / Q000737:chemistry; D005453:Fluorescence; D005583:Fourier Analysis; D006801:Humans; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D012996:Solutions; D013997:Time Factors
D004396:Coloring Agents; D012996:Solutions