A newly developed microscope-based imaging system was used to measure the oxygen tension (PO2) inside the retinal and choroidal vessels of mice and to generate in vivo maps of retinal PO2. These maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. The system was fully calibrated and used to produce retinal PO2 maps at different inspiratory oxygen fractions. PO2 rose accordingly and predictably as inspiratory O2 was stepped from hypoxic to hyperoxic conditions. Important experimental and acquisition parameters necessary for applying phosphorescence lifetime imaging to the mouse eye were investigated, including camera exposure and intensifier gain settings. Because of a need to limit light exposure to the retina, PO2 map quality as measured by the coefficient of determination was investigated as a function of signal-to-noise and accumulated excitation energy deposition. With the development of this technology for use in mice, the potential for investigating the oxygen dynamics in genetically engineered mouse models of retinal disease, including diabetic retinopathy, glaucoma, and age-related macular degeneration, is advanced.
A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.
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