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J. Reynaud, R.W. Beuerman, B. Khoobehi, J. Beach, M. Lanoue, M. Schwarz, R. Galloway-Dawkins; Confocal Hyperspectral Imaging of the Cornea . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3600.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To characterize the spectral response of the cellular and extracellular components of the cornea to narrow band incident illumination. Methods: A technique called hyperspectral imaging was used to obtain the spectral signatures of the corneal layers of an anesthetized New Zealand white rabbit. In this technique, a focal plane scanning-prism grating prism (FPS-PGP) hyperspectral camera mounted on a computer-controlled X table was interfaced to a white-light confocal microscope. Visible light (410 nm-918 nm) reflected from the sample passes through a transmission spectrometer and is imaged at the entrance slit at 256 spectral bands (2 nm resolution) to generate one image line (640 pixels wide). Multiple lines were scanned to build the entire image. The images were captured and processed to filter out background noise. The spectral characteristics of the nucleus, cytoplasm, and extracellular compartment were then extracted. Results: Reflectance from 440 nm to 730 nm was observed from the endothelial cell layer and stroma. In the near infrared part of the spectrum (730-920 nm), minimal reflectance was observed. Maximum reflectance was in the green and yellow parts of the spectrum (520-560 nm) on all layers. The endothelial cell nuclei showed the highest reflectance intensity level, followed by the cytoplasm, indicating high correlation between the cell’s spatial location and its corresponding spectral response. In the stroma a broader spectral signature was obtained for keratocyte nuclei. Conclusions: Hyperspectral imaging can be used to isolate individual cells or their structures based on their spectral signatures. It can also be used as a microscopic, non-invasive technique to identify the cell nuclei and, potentially, the state of the nucleus. In this experiment, we examined highly transparent, healthy cornea; however, other spectral characteristics such as opacification, corneal neovascularization, and cell pigmentation associated with diseased cells (i.e. tumor) could be easily detected.
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