Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen ($p_{\ce{O2}}$) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique’s utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.