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S. Notomi, T. Hisatomi, T. Kanemaru, T. Ishibashi; Observation of the Mouse Model of Choroidal Neovascularization by Fluorescent Scanning Electron Microscope (FL-SEM). Invest. Ophthalmol. Vis. Sci. 2010;51(13):5765.
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We newly created the fluorescent scanning electron microscope (FL-SEM), which is scanning electron microscope combined with fluorescent optical microscope, and stimultaneously observed both fluorescence-labeled macrophage and neovasculature and their scanning electron microscopic examination for the analysis of their localization and surficial structure in mouse model of choroidal neovascularizaion.
We made mouse model of choroidal naovascularization by 532 nm wavelength, 250 mW power, 0.1 second duration, 75 micro m spot-size laser irradiation on C57BL/6 mice. The eyes were enucleated 3, 7, 14, and 21 days after laser irradiation. After gentle removal of the retina for exposure of the surface of neovasculature, immunohistochemistry for endothelium marker CD31 and microglia / macrophage marker Iba1 were performed. Then specimens were freeze-dried and osmium-coated for the scanning electron microscopic examination. Then We observed neovasculature and surrounding microglia / macrophage by fluorescent scanning electron microscope (FL-SEM) which is scanning electron microscope combined with fluorescent optical microscope. We also observed angiogenetic-factors-positive cells and regressive process of neovascularization.
By fluorescent scanning electron microscope (FL-SEM) we observed that inflammatory cells such as microglia / macrophage infiltrate and swarm on the surface of the choroid after laser irradiation, and make network guiding choroidal neovasculature in subretinal space.
Fluorescent scanning electron microscope (FL-SEM) is a new method which have the advantage of both functional analysis by fluorescent labeling and microstructural analysis by scanning electron microscope. We can simultaneously observe the surficial structure of choroidal neovascularization and localization of infiltrating inflammatory cells. Our method enables comprehensive observation of localization of specific molecules in the three-dimensional structure. Further progression can be performed by instrumental improvement and exapansion of fluorescent labeling.
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