Fluorescence Lifetime Imaging and Application to Ca²⁺ Imaging

Abstract

Fluorescence spectroscopy is widely utilized for research in the biosciences [1–8]. These applications have been focused on two divergent disciplines, time-resolved fluorescence and fluorescence microscopy. In time-resolved measurements one takes advantage of the high information content of the time-dependent decays to uncover details about the structure and dynamics of macromolecules [4]. Such measurements are performed almost exclusively using ps laser sources coupled with high speed “single-pixel” photodetectors. While some parallel measurements have been reported, these have been for a linear array detector providing wavelength rather than spatial resolution [9]. In contrast, fluorescence microscopy is most often used to determine the localization (intensity) of the species of interest, usually of proteins or other macromolecules [6, 7]. The acquisition of two-dimensional (2D) fluorescence images is preferentially accomplished with low-speed accumulating detectors [10], which are not capable of quantifying ps-ns fluorescence decays. Consequently, the high information content of time-resolved fluorescence is not usually available for studies of microscopic biological samples. This is particularly disadvantageous when one considers the sensitivity of fluorescence decay times to chemical and environmental factors of interest, such as local pH, cation concentration, oxygen, and polarity, to name a few.