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.
Highlights
FLIM is increasingly important in microscopy because it can be used for the robust quantification of reaction kinetics [2], pH [3], molecular association [4], aggregation [5] molecular proximity [6] and Forster resonance energy transfer [7] among many other parameters
The experiment was repeated with Rose Bengal (10 μM in ethanol), and a monoexponential decay of 760 ps was measured, which is in good agreement with time correlated single photon counting (TCSPC)
Though multi-harmonic FLIM (mhFLIM) acquired the lifetime images more than 20× faster than TCSPC, the signal-to-noise ratio (SNR) achieved by each method is similar
Summary
FLIM is increasingly important in microscopy because it can be used for the robust quantification of reaction kinetics [2], pH [3], molecular association [4], aggregation [5] molecular proximity [6] and Forster resonance energy transfer [7] among many other parameters. There are several techniques for performing FLIM; here we focus on widefield FD-FLIM, which provides the key benefits of rapid measurement capability and comparatively high photon efficiency. The majority of FD-FLIM systems are restricted to the measurement of single exponential lifetime decays. Monoexponential decays are the exception rather than the rule in biological imaging, as each pixel in the image may contain multiple fluorophores, with different fractional concentrations, and often different local environments. The fluorophore itself may exhibit inherently biexponential fluorescence decays. In this case, an analysis assuming a monoexponential response will be qualitative at best, and often misleading [8]. A simple, robust and efficient method for extending the capability of FD-FLIM systems to measure multiple lifetime components simultaneously is a highly desirable objective
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