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R.D. Shonat, K.M. Hawkins; Choroidal and Retinal Oxygen Tenison Imaging: Towards a Three-Dimensional Representation of Oxygen in the Vessels that Supply the Rodent Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4873.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Insufficient oxygen delivery and retinal hypoxia have been implicated as causal for the development of many devastating diseases of the eye, including diabetic retinopathy and age-related macular degeneration. While the two-dimensional imaging of oxygen tension (PO2), based on phosphorescence quenching, has now been applied in a variety of different animal models, it is fundamentally a luminescence-based system lacking depth discrimination. The separate contribution of the choroidal and retinal vasculatures to the state of retinal oxygenation has never been rigorously addressed. Such a separation would significantly advance our understanding of oxygen delivery dynamics in these two very distinct vasculatures. In this study, we investigate a new strategy for optical sectioning of PO2 maps in the rodent retina using our current phosphorescence-based lifetime imaging system. Methods: Phase-sensitive phosphorescence image sets (15 images per set) were generated using oxygen-sensitive probe excitation with both blue (λ = 416 nm) and green (λ = 524 nm) light. The mathematical algorithms needed to convert phase-sensitive image sets into PO2 maps were modified to analyze both image sets simultaneously and to extract two phase shift estimates at each pixel. With these two phase-shift maps, two distinct oxygen tension maps could be generated. Mathematical estimates of excitation light penetration depth were used to assist the curve-fitting process. Results: Image sets generated with blue and green excitation light were substantially different, reflecting the differences in light penetration depth. The simultaneous analysis of the two image sets using the modified fitting algorithms resulted in the generation of two PO2 maps: one retinal and the other choroidal. Efficient separation of these two vasculatures was straightforward in regions dominated by large retinal vessels, but more problematic in regions dominated by capillaries. The effect of increasing the number of images per set and further enhancements to the fitting algorithms is currently being investigated to address this problem. Conclusions: A new imaging strategy that uses two separate excitation wavelengths and extends the mathematical algorithms to permit the determination of two distinct phase shifts and the generation of both retinal and choroidal PO2 maps is described. The ability to discriminate between the two different vasculatures that bathe the retinal with oxygen is of critical importance to our understanding of oxygen dynamics in retinal disease.
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