Computational protocol: Two-Dimensional Plane for Multi-Scale Quantification of Corneal Subbasal Nerve Tortuosity

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Protocol publication

[…] One hundred forty de-identified images of the subbasal nerve plexus of the central cornea were selected from an existing database. Images were acquired from 76 subjects (49 women and 27 men, 54.9 ± 19.3 years old) with laser scanning IVCM (Heidelberg Retina Tomograph 3 with the Rostock Cornea Module; Heidelberg Engineering GmbH, Heidelberg, Germany). The diode laser has a 670-nm red wavelength, and the microscope is equipped with a ×63 objective lens with a numerical aperture of 0.9 (Olympus, Tokyo, Japan). The images obtained represent a coronal section of the cornea of 400 × 400 μm2, from the subbasal layer. For imaging, a disposable sterile polymethylmethacrylate cap (Tomo-Cap; Heidelberg Engineering) was filled with a layer of hypromellose 0.3% gel (GenTeal; Alcon, Fort Worth, TX, USA) and mounted on the Cornea Module. After topical anesthesia with 0.5% proparacaine hydrochloride (Alcon), a drop of hypromellose 0.3% gel was applied to both eyes. One drop of this gel was also put on the outside tip of the cap to improve optical coupling. Then, the Cornea Module of the confocal microscope was advanced until the gel contacted the central surface of the cornea. Digital images were then recorded at a rate of 3 frames per second in sequence mode, including 100 images per sequence, from all layers of the cornea.For this pilot-level study, 140 images showing a wide range of tortuosity characteristics were chosen from patients with a variety of eye conditions, typically at a depth of 50 to 80 μm (). Specifically, 20 images were taken from healthy subjects, 100 images from patients with dry eye disease, and 20 images from subjects with herpes simplex keratitis. Images were graded independently by three expert observers (PH, AK, and SA) using four tortuosity levels according to a previously published protocol. Nerves were traced using NeuronJ (in the public domain at, a plug-in for the ImageJ software (in the public domain at study was performed in accordance with the tenets of the Declaration of Helsinki. […]

Pipeline specifications

Software tools NeuronJ, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Papilloma, Choroid Plexus