Two-photon-based phosphorescence lifetime imaging (2P-PLIM) for in vivo oxygen measurements at high spatial and temporal resolution
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Phosphorescence lifetime imaging has been gaining popularity in the two-photon microscopy world, attributed to the development of two-photon-excitable oxygen-sensing probes [1, 2]. Lifetime of their phosphorescence is in the microsecond range and shortened in presence of molecular oxygen (‘oxygen-dependent quenching of phosphorescence’). Two-photon-based phosphorescence lifetime imaging microscopy (2P-PLIM) enables quantitative estimation of the oxygen concentration – as partial pressure of oxygen (pO2) – within a given tissue in vivo at high spatial resolution. Rapid and frequent phosphorescence lifetime acquisition cycles supported through microscope hard- and software allow users to perform functional measurements at high temporal resolution.
We use 2P-PLIM to measure pO2 in the brain of anesthetized and awake rodents [3, 4] aiming to understand the intricate balance between neuronal activity-driven oxygen consumption and oxygen delivery from the blood, which is the underlying mechanism of human neuroimaging studies based on functional MRI . However, 2P-PLIM can also be used in areas other than neurosciences, e.g., to study tumor microenvironment, inflammation, and other physiological or pathological processes in vivo.
Here, we show tissue pO2 measurements with Oxyphor 2P  in the somatosensory cortex of awake head-fixed mice. Oxyphor 2P was microinjected through a silicone port below a cranial window. 2P-PLIM was performed with a Bruker Ultima 2-photon LSM equipped with a GaAs photomultiplier module connected to a time-correlated single photon counting card integrated into the microscope’s hard- and software. Tissue pO2 was measured in vicinity of a ‘diving’ arteriole, a small blood vessel that delivers oxygen-rich blood from the brain surface to deeper cortical layers. As expected, we observed spatial pO2 gradients around the diving arteriole, from high pO2 close to the arteriole to lower pO2 further away from the arteriole [2, 3]. Currently, we use these measurements to estimate laminar profiles of and cell-type-specific contributions to oxygen consumption in cerebral cortex.
 Finikova, et al., ChemPhysChem 2008, 9(12):1673-79. DOI: 10.1002/cphc.200800296.
 Esipova, et al., Cell Metabolism, 2019, 29(3):736-744. DOI: 10.1016/j.cmet.2018.12.022.
 Sakadžić, et al., Nature Methods 2010, 7(9):755-759. DOI:10.1038/nmeth.1490.
 Sakadžić, et al., Neurophotonics 2016, 3(4). DOI: 10.1117/1.NPh.3.4.045005.
 Uhlirova, et al., Philosophical Transactions of the Royal Society B: Biological Sciences 2016, 371(1705):20150356. DOI: 10.1098/rstb.2015.0356.