mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy

Opt Express. 2009 Feb 2;17(3):1557-70. doi: 10.1364/oe.17.001557.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Calibration
  • Cell Line, Tumor
  • Coloring Agents / chemistry
  • Fluorescence
  • Fourier Analysis
  • Humans
  • Microscopy / instrumentation*
  • Microscopy / methods*
  • Solutions
  • Time Factors

Substances

  • Coloring Agents
  • Solutions