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X. Huang, V. Shestopalov, R.W. Knighton; Measuring Retinal Nerve Fiber Bundle Thickness in Previously Imaged Tissue . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5507.
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Purpose: In order to relate optical measurements of the retinal nerve fiber layer (RNFL) to the underlying structure, one must have accurate values for RNFL thickness at the locations measured optically. The purpose of this study was to develop a method for measuring RNFL thickness at any location on retinal tissue previously studied by imaging reflectometry or polarimetry. Methods: The method developed used confocal laser scanning microscopy (cLSM) to provide both en face and cross–sectional images of a whole mounted retina. Nerve fiber bundles were identified by using phalloidin to label actin and ganglion cell bodies were identified by DAPI fluorescent counterstain of nuclei. Isolated rat retina prepared as for imaging micropolarimetry was fixed with 4% paraformaldehyde, followed by standard procedures of DAPI and phalloidin staining. The retina flat–mounted onto slides was examined by cLSM. 2–D images were collected through the retina to a depth at least covering the ganglion cell layer. The images were stacked to reconstruct the measured retina in 3–D. Cross–sectional images were synthesized from the reconstruction. Results: The en face images displayed individual nerve fiber bundles and ganglion cells between bundles (Fig. A, 144 µm × 144 µm). Blood vessels, which bound much more phalloidin, were easily distinguished from nerve fiber bundles (BV in Fig. A). In cross–sectional images, a single layer of ganglion cells lying under the RNFL was seen (Fig. B, 144 µm × 29 µm). Thickness of nerve fiber bundles could be measured on any cross section of the bundles (Fig. B) or along a bundle (Fig. C) with an accuracy of 1 µm. Conclusions: Retinal nerve fiber bundles in a whole mounted retina were identified with phalloidin. The en face image displayed the same pattern of nerve fiber bundles and blood vessels as seen in imaging measurements and simplified the identification of corresponding areas in the two modalities. The cross–sectional images provided thickness measurements of the RNFL over the entire field–of–view, not just at the points represented by a conventional histologic section, resulting in a large increase in available data. View OriginalDownload SlideView OriginalDownload Slide
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