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Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue


Measurements of oxygen partial pressure (pO2) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO2 measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here we report to our knowledge the first practical in vivo two-photon high-resolution pO2 measurements in small rodents' cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon–enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 μm, sub-second temporal resolution and requires low probe concentration. The properties of the probe allowed for direct high-resolution measurement of cortical extravascular (tissue) pO2, opening many possibilities for functional metabolic brain studies.

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Figure 1: Phosphorescence detection procedure.
Figure 2: Measurement of pO2 in cortical microvasculature.
Figure 3: Measurement of pO2 in cortical tissue.
Figure 4: Simultaneous measurement of pO2 in cortical vasculature and tissue.


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We thank W. Wu for performing the rat surgery, C. Ayata and G. Boas for critically reading the manuscript and support from US National Institutes of Health grants R01NS057476, P50NS010828, P01NS055104, R01EB000790, K99NS067050, R01HL081273 and R01EB007279 and American Heart Association grant 0855772D.

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Authors and Affiliations



S.S., M.A.Y. and V.J.S. designed the microscope. E.R. synthesized the probe. S.S., M.A.Y., E.T.M., A.D., K.A. and S.R. performed experiments. S.S., D.A.B. and S.A.V. analyzed the data. D.A.B., S.A.V., E.H.L., S.S. and A.D. conceptualized and directed the research project. All authors discussed the results and commented on the manuscript.

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Correspondence to Sergei A Vinogradov or David A Boas.

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The authors declare no competing financial interests.

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Sakadžić, S., Roussakis, E., Yaseen, M. et al. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue. Nat Methods 7, 755–759 (2010).

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