Better phasor calibration in turbid media using Monte Carlo simulations.

Abstract

Phasor analysis has gained acceptance as a simpler yet quantitative alternative to nonlinear fitting approaches for in vitro fluorescence lifetime imaging microscopy (FLIM) and is incorporated into many commercial and research analysis software. FLIM typically involves cell monolayers, thin tissue slices or transparent spheroids, which do not introduce significant light scattering or absorption effects. As a result, the recorded fluorescence decay is a mere convolution product of the instrument response function and the emitted decay. However, the situation is more complex when in tissue phantoms or live animals observed with macroscopic fluorescence lifetime imaging (MFLI) systems, where those effects become noticeable. In particular, when using the phasor approach to analyze MFLI data, they result in an inadequacy to properly calibrate the phasor of measured decays using the simple approach used in FLIM. Namely, a discrepancy between observed calibrated phasor locations and those expected is generally observed in the simple cases where single-exponential decays are known to occur, no matter what calibration approach is used. This calls for a way to take these effects into account to obtain more quantitative results in this general situation of fluorescence in turbid media.