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M.M. Campos, R.N. Fariss, W.G. Robison, Jr.; Novel Techniques For Imaging Retinal Vessels Using Whole Retinas After Long Term Fixation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2416.
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Purpose: Aldehyde–based fixatives (e.g., 4% paraformaldehyde in PBS, 10% buffered formalin, 2–4% buffered glutaraldehyde) provide a convenient and reliable method for preserving tissues for extend periods of time. A major disadvantage of long–term aldehyde fixation is background autofluorescence, a characteristic that increases with time. High levels of autofluorescence often complicate epifluorescence microscopy. This study utilized procedures that took advantage of the high levels of fixation–induced autofluorescence in intact human and rat retinas that had been stored in aldehyde fixatives for up to 17 years. Methods: Eyes were opened near the equator, the retina was carefully removed, the vitreous was brushed off, and the whole retina was mounted on a slide. A Leica SP2 laser scanning confocal microscope was utilized for precise characterization of the autofluorescence emission spectra and identification of the regions within these spectra that yielded optimal contrast between vascular and non–vascular tissues. Results: Collection of thin optical sections under conditions of optimal contrast, permitted high resolution imaging of vascular structures from "unstained" archival specimens. Fresh and lightly fixed retinas did not provide equivalent contrast or clear tissue distinction. Conclusions: These novel techniques have permitted, utilization of background autofluorescence to visualize minute structural features of retinal vessels, identification and quantification of normal and pathological vascular features in unstained archival samples, preservation of complex 3D vascular networks and localization of vascular lesions to specific retinal layers.Thus, the increased autofluorescence arising from long–term fixation that would otherwise cause unwanted background signal became an asset.
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