An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure . The instrument was demonstrated by performing depth-resolved microvascular measurements of rat cortical vessels down to below the surface. 2P excitation of porphyrin was confirmed, and measured values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only measurement techniques.
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