Fluorescence lifetime imaging microscopy: Pixel-by-pixel analysis of phase-modulation data

Abstract

The novel techniques of fluorescence lifetime imaging (FLI) and FLI-microscopy (FLIM) in the frequency domain enable the mapping of the spatial distribution of fluorescence lifetimes of a specimen. The analysis of FLI(M) data is described. Two computer programs were developed to determine nanosecond fluorescence lifetimes from digitized phase-resolved images produced by the FLI(M) instrument, for single- and double-component systems, respectively. Image processing routines were incorporated for optimal display of the lifetime analysis. The combination of analysis and image processing yields images of the fluorescence lifetimes and of the associated analytical and statistical parameters. The error analysis enables the estimation of the quality of the FLI(M) data at each pixel of the image. The features of the programs are illustrated by simulated and experimental FLIM data, generated in the latter case with commercially available fluorescence microspheres as references for spatial and temporal resolution.