Fluorescence Lifetime Imaging Microscopy (FLIM) Data Analysis with TIMP

Abstract

Fluorescence Lifetime Imaging Microscopy (FLIM) allows fluorescence lifetime images of biological objects to be collected at 250 nm spatial resolution and at (sub-)nanosecond temporal resolution. Often n_comp kinetic processes underlie the observed fluorescence at all locations, but the intensity of the fluorescence associated with each process varies per-location, i.e., per-pixel imaged. Then the statistical challenge is global analysis of the image: use of the fluorescence decay in time at all locations to estimate the n_comp lifetimes associated with the kinetic processes, as well as the amplitude of each kinetic process at each location. Given that typical FLIM images represent on the order of 10² timepoints and 10³ locations, meeting this challenge is computationally intensive. Here the utility of the TIMP package for R to solve parameter estimation problems arising in FLIM image analysis is demonstrated. Case studies on simulated and real data evidence the applicability of the partitioned variable projection algorithm implemented in TIMP to the problem domain, and showcase options included in the package for the visual validation of models for FLIM data.