O-2 PLIM microscopy was employed in various studies, however current platforms have limitations in sensitivity, image acquisition speed, accuracy and general usability. We describe a new PLIM imager based on the Timepix3 camera (Tpx3cam) and its application for imaging of O-2 concentration in various tissue samples stained with a nanoparticle based probe, NanO(2)-IR. Upon passive staining of mouse brain, lung or intestinal tissue surface with minute quantities of NanO(2)-IR or by microinjecting the probe into the lumen of small or large intestine fragments, robust phosphorescence intensity and lifetime signals were produced, which allow mapping of O-2 in the tissue within 20 s. Inhibition of tissue respiration or limitation of O-2 diffusion to tissue produced the anticipated increases or decreases in O-2 levels, respectively. The difference in O-2 concentration between the colonic lumen and air-exposed serosal surface was around 140 mu M. Furthermore, subcutaneous injection of 5 mu g of the probe in intact organs (a paw or tail of sacrificed mice) enabled efficient O-2 imaging at tissue depths of up to 0.5 mm. Overall, the PLIM imager holds promise for metabolic imaging studies with various ex vivo models of animal tissue, and also for use in live animals.