Computational protocol: Wide-field corneal subbasal nerve plexus mosaics in age-controlled healthy and type 2 diabetes populations

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Protocol publication

[…] Nerve fibers visible in all mosaics were traced using two approaches: manual and fully automated. An experienced observer manually traced the centerlines of all visible nerves using the NeuronJ tracing plugin for ImageJ (publicly available at in the acquired images, as previously described. The resulting manual tracings were checked for consistency and accuracy by a second, independent experienced grader, and anomalies and missed nerves in the original manual tracings were corrected. The total length of all traced nerves was determined in NeuronJ in millimeters, and this value was divided by the mosaic area in mm2, yielding the mosaic nerve fiber length density (mCNFL) in mm/mm2 (Data Citation 1).Automatic nerve tracing was based on a previously developed algorithm, but applied here to large-scale mosaics for the first time. Briefly, corneal nerve visibility was initially enhanced using a bank of log-Gabor filters. The resulting filtered image was then thresholded to obtain candidate nerve segments (all string-like structures present in the image). Finally, these candidate nerve segments were classified as nerves or 'other' using a Support Vector Machines (SVM) approach. The length of all traced segments was then automatically determined, and divided by the mosaic area to yield mCNFL in mm/mm2 (Data Citation 1). Specifically for mosaics, to avoid underestimation bias due to mosaic edge effects (where no nerves are visible due to partial oblique imaging of the corneal epithelium or Bowman’s layer), the mosaic area used for the nerve length density calculation excluded these edge regions containing anatomic layers outside the SBP (for both manual and automatic approaches).To demonstrate the possibility to individually analyze specific regions of the SBP, nerve length density within the infero-central whorl,, region (wCNFL) was computed. An expert identified the center position (origin) of the whorl region in each mosaic containing an identifiable whorl. Then a circular region with 800 μm diameter and centered on the origin was defined. Nerve length density within this region was computed where full image data was available (Data Citation 1).All mosaics were computer-analyzed and investigators at the site where nerve tracing and quantification was performed (University of Padova) were masked to the identity of subjects and subject groups. […]

Pipeline specifications

Software tools NeuronJ, ImageJ
Application Microscopic phenotype analysis
Diseases Diabetes Mellitus, Papilloma, Choroid Plexus, Corneal Injuries