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 ([O<sub>2</sub>]), calcium ([Ca<sup>2+</sup>]), 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 singlephoton 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.
Fluorescence lifetime imaging microscopy is a technique in which the fluorescence lifetime(s) of a fluorophore
is measured at each spatially resolvable element of a microscope image. Imaging of fluorescence lifetimes enables
biochemical reactions to be followed at each microscopically resolvable location within the cell. FLIM has thus become
very useful for biomedical tissue imaging. Global analysis  is a method of recovering fluorescence decay parameters
from either time-resolved emission spectra to yield Decay-Associated Spectra , or equivalently, from FLIM datasets
to yield Decay-Associated Images. Global analysis offers a sensitive and non-invasive probe of metabolic state of
intracellular molecules such as NADH. Using prior information, such as the spatial invariance of the lifetime of each
fluorescent species in the image, to better refine the relevant parameters, global analysis can recover lifetimes and
amplitudes more accurately than traditional pixel-by-pixel analysis. Here, we explain a method to analyze FLIM data so
that more accurate lifetimes and DAIs can be computed in a reasonable time. This approach involves coupling an
iterative global analysis with linear algebraic operations. It can be successfully applied to image, e.g. metabolic states of
live cardiac myocytes, etc.