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O. Stachs, S. Knappe, J. Stave, R. Guthoff; In vivo Three–Dimensional Confocal Laser Scanning Microscopy of the Corneal Nerve Structure . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2742.
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Purpose:Evaluation of a technology for in vivo visualization of distribution and morphology of corneal nerves by means of 3D confocal laser scanning microscopy (3D–CLSM). Methods: The anterior cornea of four human volunteers was examined by an in–house developed confocal laser scanning microscope based on a commercial available instrument (Heidelberg Retina Tomograph II, Heidelberg Engineering GmbH, Germany). Raw stacks were converted using ImageJ (NIH, USA) for 3D–reconstruction using AMIRA 3.1 (TGS Inc, USA). Results: The spatial arrangement of epithelium, nerves and keratocytes was visualized by in vivo 3D–CLSM. After 3D–reconstruction of volunteers corneae volume rendering and selective oblique sections have been done to demonstrate the nerves in the central human cornea. 3D–imaging shows thick nerve bundles rising out of the deeper stroma. Main trunks which have a diameter of 12 ± 4 microns divide and spread into the subbasal plexus. Nerves further divide di– and trichotomously, resulting in fibers that are arranged parallel to Bowman's layer and are partly interconnected. The diameter of the individual nerve fibres in the subbasal plexus varies between 1.0 and 5.0 microns. The lower value is limited by the resolution of the instrument. Branches penetrating the anterior epithelial cell layer can not be visualized. Conclusions: 3D–CLSM allows in vivo visualization and analysis of the spatial arrangement of the epithelium, nerves and keratocytes of the human cornea. The developed method provides a basis for further studies of alterations of the cellular arrangement and epithelial innervation in corneal diseases. This may help to clarify gross variations of nerve fibre patterns under various clinical and experimental conditions.
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