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Gregory H. Grossman, Zhihong Chen, Bruce D. Trapp, Stephanie A. Hagstrom; In Situ Trans-scleral Imaging of the Murine Retina with Multiphoton Excitation Fluorescence Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4984.
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In an effort to develop high resolution noninvasive in vivo imaging of mouse retinas, we performed in situ trans-scleral multiphoton fluorescence microscopy (MFM) on mouse eyes.
Adult wild-type mice were given a lethal dose of ketamine/xylazine. A customized plate with a circular imaging window was placed over the center of the eye and secured to the surface of the exposed skull. The plate was then mounted on an adjustable steel frame. To expose the scleral surface for imaging, a bridle suture was placed through the corneal surface and secured to the frame, such that the eye was gently pulled to the temporal side. The imaging window of the eye mount was filled with normal saline. Trans-scleral imaging was accomplished by MFM with a 25x water emersion objective using a 730-nm femtosecond laser. In a separate analysis, enucleated intact eyes of Tie2-GFP transgenic mice were used to visualize deeper layers of the retina. These transgenic mice have Tie2 promoter-driven GFP expression resulting in specific labeling of vascular endothelial cells. Imaging of the eyes was accomplished with a 920-nm femtosecond laser to excite GFP.
Complied Z axis-stacks obtained from in situ imaging revealed hexagonal cells of the retinal pigment epithelium (RPE), most likely due to autofluorescence of retinosomes within RPE cells. GFP-labeled vessels of the enucleated eyes were seen at two distinct levels. The choroidal plexus was imaged superficially to the RPE, followed by a network of vessels likely representing the retinal vasculature.
Taking advantage of endogenous fluorophores, trans-scleral MFM has the potential to be a useful non-invasive in vivo imaging modality for the RPE and could be used to monitor disease states in mouse models of human retinal disorders. Our results also show that MFM has the ability to image structures in the neural retina, opening the possibility of using photoexcitable dyes to study photoreceptor physiological processes in vivo.
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