Two-photon-based Phosphorescence Lifetime Imaging (2P-PLIM) for in Vivo Oxygen Measurements at High Spatial and Temporal Resolution

Phosphorescence lifetime imaging has been gaining popularity in the two-photon microscopy world and is attributed to the development of two-photon-excitable oxygen-sensing probes [1, 2]. The lifetime of their phosphorescence is in the microsecond range and is shortened in the presence of molecular oxygen, a process called "oxygen-dependent quenching of phosphorescence". Two-photon-based phosphorescence lifetime imaging microscopy (2P-PLIM) enables the quantitative estimation of oxygen concentration as the partial pressure of oxygen (pO₂) within a given tissue in vivo and at a 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 resolutions.

We use 2P-PLIM to measure pO₂ in the brain of anesthetized and awake rodents [3, 4] aiming to understand the intricate balances at play. Specifically, 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 [5]. 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 [2] 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 laser scanning microscope equipped with a GaAs photomultiplier module that was connected to a time-correlated single-photon counting card integrated into the microscope’s hard- and software. Tissue pO₂ was measured in the vicinity of a "diving" arteriole which is 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 cell-type-specific contributions to oxygen consumption in the cerebral cortex.

[1] Finikova, et al., ChemPhysChem 2008, 9(12):1673-79. DOI: 10.1002/cphc.200800296.

[2] Esipova, et al., Cell Metabolism, 2019, 29(3):736-744. DOI: 10.1016/j.cmet.2018.12.022.

[3] Sakadžić, et al., Nature Methods 2010, 7(9):755-759. DOI:10.1038/nmeth.1490.

[4] Sakadžić, et al., Neurophotonics 2016, 3(4). DOI: 10.1117/1.NPh.3.4.045005.

[5] Uhlirova, et al., Philosophical Transactions of the Royal Society B: Biological Sciences 2016, 371(1705):20150356. DOI: 10.1098/rstb.2015.0356.

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