Global analysis of fluorescence lifetime imaging microscopy data

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the li fetime invariant fit method, each image in the lifetime image sequence is s patially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional co ntribution to the steady-state fluorescence on a pixel-by-pixel basis for e ach species. The second, superior, approach uses a global analysis techniqu e that simultaneously fits the fractional contributions in all pixels and t he spatially invariant lifetimes. In frequency domain FLIM the maximum numb er of lifetimes that can be fit with the global analysis method is twice th e number of lifetimes that can be fit with conventional approaches. As a re sult, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to sep arate the cellular distributions of coexpressed green fluorescent proteins in living cells.