Global analysis and Decay Associated Images (DAI) derived from Fluorescence Lifetime Imaging Microscopy (FLIM)

The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O₂]), calcium ([Ca²⁺]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated single-photon counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.