Analysis of time-domain fluorescence measurements using least-squares deconvolution

A main challenge in the analysis of fluorescence lifetime measurements is the ability to deconvolve the experimental data to obtain results that are free of distortions introduced by the instrument used in the experiment. The purpose of deconvolution is to free the fluorescence emission decay transients from the main sources of distortion. For time-domain fluorescence measurements, this includes distortions due to the finite rise time, width, and decay of the laser excitation system; distortions introduced by the limited frequency response of detector electronics; and distortions introduced by the light dispersion in optical fiber when fiber-optic probes are used for exciting and collecting the fluorescence emission. This chapter describes the use of the least-squares method as means of deconvolving the intrinsic fluorescence impulse response function (fIRF) from the measured fluorescence pulse transients.