Abstract
We discuss a Hadamard-transform-based fluorescence-lifetime-imaging (HT-FLI) technique for fluorescence-lifetime-imaging microscopy (FLIM). The HT-FLI uses a Fourier-transform phase-modulation fluorometer (FT-PMF) for fluorescence-lifetime measurements, where the modulation frequency of the excitation light is swept linearly in frequency from zero to a specific maximum during a fixed duration of time. Thereafter, fluorescence lifetimes are derived through Fourier transforms for the fluorescence and reference waveforms. The FT-PMF enables the analysis of multi-component samples simultaneously. HT imaging uses electronic exchange of HT illumination mask patterns, and a high-speed, high-sensitivity photomultiplier, to eliminate frame-rate issues that accompany two-dimensional image detectors.
Highlights
Fluorescence lifetimes are critical parameters for discriminating between two samples that have similar excitation and emission spectra
Thereafter, fluorescence lifetimes are derived through Fourier transforms for the fluorescence and reference waveforms
The Fourier-transform phase-modulation fluorometer (FT-PMF) enables the analysis of multi-component samples simultaneously
Summary
Fluorescence lifetimes are critical parameters for discriminating between two samples that have similar excitation and emission spectra. One way to overcome this problem is to use several time gates for each excitation [8] This is difficult for multi-component samples because the fluorescence decays need to be single exponentials. One efficient method is to prepare four time gates during one modulation period, each phase-shifted in turn by π/2 [10] Another uses a mode-locked laser and a gated, intensified CCD camera [11] to acquire "lifetime-selective" fluorescence images. (plural modulation frequencies should be used to verify single-component samples.) it is difficult to detect short-lived fluorescent molecules that have low quantum efficiencies. HT-FLI enables us to obtain fluorescence-lifetime images for every component in a sample. The combined HT-FLI system will be explained
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