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. 2020 Jan 10;10:126. doi: 10.1038/s41598-019-57022-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Counting retinal ganglion cells. (A) Raw image of Brn3a-labelled cells in a healthy rat retina. The image is a maximum-intensity projection of a stack of images collected along the z (depth) axis. Scale bar is 100 μm. (B) Image after applying a custom filter sequence and binary threshold to highlight labeled cells and a watershed function to separate overlapped cells. (C) Cells that were counted (blue) in the image using a size constraint of 15 to 300 µm². (D) Image of a flat-mounted rat retina. Dashed circles subdivide the tissue into: (1) central, (2) inner peripheral, (3) mid peripheral, and (4) outer peripheral regions within which cell counts were tabulated. Circle radii correspond to 1.9, 2.8, and 3.7 mm in retinal space. Solid line outlines the tissue border for purpose of area measurement and cell density calculation.