Quantitation of cerebral oxygen tension using phasor analysis and phosphorescent lifetime imaging microscopy (PLIM)

Abstract

Fluorescence lifetime imaging microscopy (FLIM) and phosphorescence lifetime imaging microscopy (PLIM) often require complex and computationally intensive processes for analysis. For time-domain based measurements, computation of fluorescence and phosphorescence lifetimes conventionally involves nonlinear curve fitting techniques to model the time-resolved profiles as mono- or multi-exponential decays. The phasor, or “polar plot”, analysis method has recently gained attention as a simple method to characterize variations in fluorescence lifetime. The technique involves calculations of the intensity-normalized Fourier transform of the fluorescence profiles. The phasor can be visualized by plotting the real and imaginary components on a 2-dimensional plot. We have adapted the phasor analysis method for absolute quantitation of phosphorescence lifetimes of oxygen-sensitive phosphors. We utilize the phasor-derived lifetime values to quantify oxygen partial pressure in cortical micro vessels of awake mice. Here, we describe the modifications to adapt the technique for longer-duration phosphorescence decays. Our results demonstrate that oxygen measurements obtained from phasor analysis are in strong agreement with traditional curve fitting calculations. Using simulated phosphorescence decays, we also compare the effectiveness of the phasor method to nonlinear curve fitting techniques. To our knowledge, these findings constitute the first application of the phasor analysis method for characterizing phosphorescence measurements on the microsecond time scale. The method shows promise for monitoring cerebral metabolism and pathological changes in preclinical rodent models.