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Matthew Petroll, Daniela Hagenasr, H Cavanagh, Danielle Robertson; 3-Dimensional Assessment of In Vivo Corneal Wound Healing using a Modified HRT-RCM Confocal Microscope. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3222. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We previously described hardware and software modifications for the Heidelberg Engineering Rostock Corneal Module (HRT-RCM) that allowed quantitative 3-D scanning of the full thickness cornea in vivo. The purpose of this study was to test the capabilities of this system for assessing corneal sub-layer thicknesses as well as keratocyte backscattering, morphology and connectivity following a full-thickness corneal injury.
In order to test the HRT system for quantitative imaging during wound healing, rabbit corneas were scanned both before and 7, 14 and 28 days after transcorneal freeze injury (FI), which damages all corneal cell layers. To automate the HRT-RCM focusing mechanism, a PC-controlled motor drive was attached to the microscope housing. Continuous scans were made from the endothelium to the epithelium at a constant lens speed of 60 μm/sec, while collecting images at a rate of 30 frames/second using HRT streaming software (beta software). Image sequences were decoded to allow depth calculation and measurement of sub-layer thicknesses. Estimates of corneal backscattering were obtained by measuring the area under intensity vs. depth curves. In some corneas, image stacks were aligned using NIH Image, and 3-dimensional reconstructions were generated using Imaris.
Using the modified HRT-RCM, full thickness CMTF scans with clear images of all cell layers were consistently obtained. FI induced damage to the epithelial and endothelial cell layers, which led to significant stromal edema at 7 days (as indicated by increased thickness). Edema partially subsided at 14 and 28 days as these cell layers were re-established. When keratocytes were in their quiescent state, only the cell nuclei were visible by confocal microscopy. Following FI, stromal cells repopulating the damaged tissue assumed an elongated and interconnected fibroblastic morphology, and a significant increase in cellular light scattering was measured. This stromal haze gradually decreased as wound healing progressed.
Overall, this modified system allows high resolution 3-D image stacks from the full thickness rabbit cornea to be obtained during in vivo wound healing. These datasets can be used for interactive visualization of corneal cell layers, measurement of sub-layer thickness, assessment of cell morphology and connectivity, and estimation of stromal backscatter (haze) during wound healing.
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