Fully automated detection, segmentation, and analysis of in vivo RPE single cells.

To develop a fully automated method of retinal pigmented epithelium (RPE) cells detection, segmentation and analysis based on in vivo cellular resolution images obtained with the transscleral optical phase imaging method (TOPI). Fourteen TOPI-RPE images from 11 healthy individuals were analysed. The developed image processing method encompassed image filtering and normalisation, detection and removal of blood vessels, cell detection and cell membrane segmentation. The produced measures were cellular density of RPE layer, cell area, number of neighbouring cells, eccentricity, circularity and solidity. In addition, we proposed coefficient of variation (CV) of RPE cellular membrane (CMD <sub>CV</sub> ) and the solidity of the RPE cell membrane-shape as new metrics for the assessment of RPE single cells. The observed median cellular density of the RPE layer was 3743 cells/µm <sup>2</sup> (interquartile rate (IQR) 1687), with a median observed RPE cell area of 193 µm <sup>2</sup> (IQR 141). The mean number of neighbouring cells was 5.22 (standard deviation (SD) 0.05) per RPE cell. The mean RPE cell eccentricity was 0.67 (SD 0.02), median circularity 0.83 (IQR 0.01), and median solidity 0.92 (IQR 0.00). The median CMD <sub>CV</sub> was 0.19 (IQR 0.02). The method is characterised by a median image processing and analysis time of 48 sec (IQR 12) per image. The present study provides the first fully automated quantitative assessment of human RPE single cells in vivo. The method provides a baseline for future research in the field of clinical ophthalmology, enabling characterisation and diagnostics of retinal diseases at the single-cell level