An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and pO(2) quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure pO(2). The instrument was demonstrated by performing depth-resolved microvascular pO(2) measurements of rat cortical vessels down to 120 microm below the surface. 2P excitation of porphyrin was confirmed, and measured pO(2) values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D pO(2) measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only pO(2) measurement techniques.