A semi-automated assessment of cell size and shape in monolayers, with optional adjustment for the cell–cell border width—application to human corneal endothelium

Abstract

Measurements of large numbers of feature sizes within defined domains (e.g. cell areas within cell–cell borders) can be a time-consuming activity, but automation that includes defining such domains has not be proven to be very reliable. Other alternatives are therefore needed, and the goal of the present studies was both to develop a semi-automated (interactive) measurement system for cell areas and to carefully compare the output to that obtained using a manual digitiser pad method. A particular interest was in the contribution made by the cell–cell border zones. Non-contact specular micrographs of central corneal endothelium were obtained from 20 white male adults, aged 40–60 years. An overlay of the endothelial image was generated manually, from which the areas of around 200 cells were measured manually with a digitiser pad and also by a computer-assisted scanning method. The pad data was typed into a spread sheet along with details of the number of cell apices (sides). The computerised analysis identified borders of the same cells on the overlay, reduced these borders to a minimum, and then assessed cell area by the pixel count along with the number of neighbouring cells (to give cell sides data). The average cell area was 393±28 and 422±29 μm 2 (mean±SD) by the digitiser pad and computer-based methods, respectively. The average areas for each cell type were 153, 270, 392, 519 and 685 μm 2 for 4-, 5-, 6-, 7- and 8-sided cells, respectively. Assessment of the relationship between cell area and the number of cell sides (area–side relationships) showed a highly significant and positive correlation ( P<0.001; r 2=0.865). Comparing the two methods, the average cell area was 7.5% higher in the computer scan method, and this is attributed to the fact that the contribution made by the cell borders (the para-cellular space) had been essentially eliminated. A proportional correction factor can be applied to add back the cell borders/intercellular space to the computerised output, and examples are given based on using the average data from digitiser pad for each cell type. In conclusion, a computer assisted method has been developed to simultaneously provide data on the variance in cell areas (polymegethism) and cell shape (pleomorphism) from overlays of 200 cells from human corneal endothelial images, with the cell border zone corrected to allow for a finite para-cellular space.