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W. M. Petroll, Matthew Weaver, Saurabh Vaidya, James P. McCulley, H. Dwight Cavanagh; Quantitative Corneal Imaging In Vivo Using a Modified HRT- RCM Confocal Microscope. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4205.
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© ARVO (1962-2015); The Authors (2016-present)
The HRT Rostock Corneal Module (HRT-RCM) provides excellent resolution, contrast and optical sectioning capability - defining features of confocal microscopy. However, changing the focal plane over large distances must be performed manually, which limits its ability to perform quantitative 3-D imaging. We recently modified the HRT-RCM so that the focal plane position can be controlled remotely using a motorized lens drive system. The purpose of this study was to test the feasibility of performing quantitative full-thickness corneal imaging using this prototype system.
The left eyes of six New Zealand White rabbits were repeatedly scanned from endothelium to epithelium at a speed of 60 µm/sec, and images were continuously acquired using HRT streaming software. The resulting image sequence (".vol" file) was read into a custom-developed program, in which the z-position of each image was calculated based on the encoded acquisition time and lens speed. The dataset was converted into a format compatible with in-house software for interactive 3-D viewing and measurement of sub-layer thicknesses. In one cornea, following automated registration of the 3-D stacks using Image J, manual counts of keratocyte nuclei were performed using Metamorph. This cornea was also fixed in situ following sacrifice, labeled with propidium iodide, and imaged in vitro using a Leica SP2 confocal microscope.
The mean epithelial and corneal thickness measured using the modified HRT-RCM were 48.6 + 6.6 µm and 377.6 + 15.4 µm, respectively (n=6 corneas). The average coefficients of variation for repeated scans were 2.3% and 2.0%, respectively. The mean overall keratocyte density measured in vivo was 46,715 + 584 cells/mm3, which was in good agreement with the in vitro measurement of 45,975 cells/mm3. In both cases there was a gradual decrease in density from the anterior to posterior cornea, as previously reported (R = 0.83, p < 0.001).
This modified system allows high resolution 3-D image stacks to be collected from the full thickness cornea in vivo. These datasets can be used for interactive visualization of corneal cell layers, measurement of sub-layer thickness, and depth-dependent keratocyte density measurements. Overall, the modifications significantly expand the potential quantitative applications of the HRT-RCM confocal microscope.
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