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

Time-domain measurements for fluorescence lifetime imaging microscopy (FLIM) and phosphorescence lifetime imaging microscopy (PLIM) are conventionally computed by nonlinear curve fitting techniques to model the time-resolved profiles as mono- or multi-exponential decays. However, these techniques are computationally intensive and prone to fitting errors. The phasor or "polar plot" analysis method has recently gained attention as a simple method to characterize fluorescence lifetime. Here, we adapted the phasor analysis method for absolute quantitation of phosphorescence lifetimes of oxygen-sensitive phosphors and used the phasor-derived lifetime values to quantify oxygen partial pressure (pO2) in cortical microvessels of awake mice. Our results, both experimental and simulated, demonstrate that oxygen measurements obtained from computationally simpler phasor analysis agree well with traditional curve fitting calculations. To our knowledge, the current study constitutes the first application of the technique for characterizing microsecond-length, time-domain phosphorescence measurements and absolute, in vivo quantitation of a vital physiological parameter. The method shows promise for monitoring cerebral metabolism and pathological changes in preclinical rodent models.