Confocal microscopic study of rabbit and human corneal epithelia using two objectives with different numerical aperture

Abstract

Purpose: The prospective study compares the confocal microscopic images of the rabbit and human corneal epithelial layers obtained with two objectives having different numerical apertures (NA). Methods: Ten rabbit eyes were enucleated and kept in place by a pneumatic support to ensure the best performance of each objective, with no disturbance due to eye movements. Five normal patients were selected after agreement. The rabbit eyes and those of the patients were all examined with slit lamp. Confocal microscopy (Tomey, Erlangen-Tennenlohe, Germany) was performed with Achroplan (Zeiss, Oberkochen, Germany) 40x/NA = 0.75 and 63x/NA = 0.9 objectives. Selected images of the corneal epithelium layers were evaluated qualitatively for the size, shape, light scattering, and reflection of the cells. Results: The cells in the superficial corneal epithelium of rabbit and human eyes, before desquamation, produced a much brighter reflection than the surrounding cells. This was clearly seen only with the 63x/NA = 0.9 objective. The corneal epithelium subsegments of the rabbit eyes, like the basal cell layer or the intermediate cell layer (wing cells), were visible under the high numerical aperture (63x/NA = 0.9) objective only. These layers, in the human eyes, were more clearly visible using the high numerical aperture objective. Conclusion: An objective with a high numerical aperture and a high optical resolution provided an improved analysis to study all the layers of the rabbit and human corneal epithelium.