Segmentation methods and morphometry of confocal microscopy imaged corneal epithelial cells

Purpose: To develop and explore automated cell identification and segmentation methods for morphometry of confocal microscopy imaged corneal epithelial cells using ImageJ software. Methods: In vivo confocal microscopy images of the intermediate (wing) and basal cell layers of the central and peripheral corneas of 20 healthy participants were analysed. The intermediate and basal cell areas obtained using the two new techniques (i.e., manual- and auto- thresholding) were compared with the widely used manual tracing technique. A predefined range of epithelial cell morphometric parameters was used as image descriptors to improve cell identification and segmentation. Results: The mean intermediate cell area obtained using the manual tracing (central; 120 ± 14 µm², peripheral; 123 ± 15 µm²) was statistically similar (p > 0.05) to the manual thresholding (central; 119 ± 7 µm², peripheral; 119 ± 8) but not with the auto thresholding technique (central; 101 ± 8 µm², peripheral; 101 ± 7 µm²). Bland-Altman limits of agreement for the mean difference (measurement bias) in central and peripheral intermediate cell area determined via manual tracing and manual thresholding techniques were 1 µm² (+25 to − 23 µm²) and 4 µm² (+29.8 to − 21.9 µm²). There were statistically significant differences in basal cell area between the three methods. Conclusion: The manual thresholding technique may be used for automated identification and segmentation of corneal epithelial intermediate cells (central and peripheral) for assessing various morphometric parameters. However, measurement of the corneal epithelial basal cells is less reliable using thresholding techniques.