Development of a dual microscope for simultaneous observation of oxygen distribution and blood flow in the microvascular bed

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We developed an oxygen-quenching fluorescent membrane technique for in vivo visualization of pO2 distribution in the rat mesenteric microcirculation as optical patterns on the oxygen-sensitive membrane by epifluorescence microscopy. This membrane is highly gas-permeable to allow pO2 measurement, and is transparent enough to also permit observation of the microcirculation. By placing the oxygen-sensitive membrane in close proximity to the surface of mesentery without disturbing blood flow, it is possible to visualize the pO2 distribution in the microvascular network. We also developed a new microscope system for dual observation of pO2 distribution and microcirculation by using the oxygen-sensitive membrane technique.This new system is composed of two microscopes aligned face-to-face, a gastight and watertight chamber for the rat mesentery placed between two objectives, and a video recording system. One microscope (M1) equipped with a mercury lamp is employed for epi-illumination, and the other (M2) equipped with a halogen lamp for transillumination. A correcting diagonal mirror inserted in the optical path of M1 ensures proper direction of the two images. After -1 x1 mm area of the mesentery is selected through microscope M1 under transillumination, the light source is switched to the mercury lamp (B-excitation) for simultaneous observation of the fluorescence image through M1 and the microcirculation through M2. The recorded fluorescence image is digitized and converted to pO2 distribution by using Stern-Volmer relation, which can be displayd as 2-dimensional, 3-dimensional, or isobaric contour maps.The present system, which allows the simultaneous determination of pO2 distribution and microcirculatory parameters, should provide a novel approach to studies on the relationship between hemodynamics and oxygen transport dynamics under normal and abnormal conditions.