Quantitative analysis of ring-shaped (crater-like) features at the tear film–epithelial interface of the rabbit cornea as assessed by scanning electron microscopy

Purpose: To develop a method for the quantitative analyses of the ring-shaped features usually observed on the surface of mammalian corneal epithelial cells. Methods: The corneas of 10 young adult female albino rabbits were prepared for scanning electron microscopy (SEM) by fixation in 2% glutaraldehyde in 80 mM cacodylate buffer (pH 7.2 to 7.4, 320 to 340 mOsm/kg) and single images taken at 1000× of the central region of the corneal epithelial surface. On enlarged prints at 5000× final magnification, cell-cell borders were marked as were the outlines of the ring-shaped features. Cell areas and the dimensions of the ring-shaped features were measured and the cell characteristics in terms of electron reflex (light, medium, and dark) as well as the number of apices (sides) also noted. Results: The ring-shaped features had an overall density of 2.5/100 µm, or an average of 6.5/cell. With the corneal epithelial cells being small and light in appearance (median area 36 µm), medium in size and appearance (median area 151 µ m), or larger and darker (median area 476 µ m), the number of features/cell was very strongly associated with both the cell type (p < 0.001) and the cell area (p < 0.001). The ring-shaped features could be between 0.65 and 5.5 µm in diameter, but sizes more than 3 µm were most unusual. Ring-shaped features of light cells were smallest (median diameter 1.35 µ m), slightly larger on medium cells (1.55 µm), and largest on the dark cells (1.90 µm). Conclusions: The ring-like features seen on SEM photomicrographs of rabbits (also known as craters or holes) are reproducible in terms of their overall density per unit surface area as well as their diameter. Such methods should be useful for objective analysis of these features after various treatments of the ocular surface.