An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and ${\mathrm{pO}}_2$ quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure ${\mathrm{pO}}_2$ . The instrument was demonstrated by performing depth-resolved microvascular ${\mathrm{pO}}_2$ measurements of rat cortical vessels down to $120\mathrm{\mu }\mathrm{m}$ below the surface. 2P excitation of porphyrin was confirmed, and measured ${\mathrm{pO}}_2$ values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D ${\mathrm{pO}}_2$ measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only ${\mathrm{pO}}_2$ measurement techniques.