The Effect of Fixation on Corneal Endothelial Cell Dimensions and Morphology in Scanning Electron Microscopy

Abstract

Soft tissue specimens shrink during fixation, dehydration and critical point drying when prepared for scanning electron microscopy (SEM). This can cause serious artifacts not only in 'compact' tissues but especially in hollow structure, like the eye, where the chambers are lined by delicate layers such as the corneal endothelium. In this study various glutaraldehyde and formaldehyde fixations at different concentrations with or without 5% sucrose were tested. Dimensional as well as morphological changes of rabbit corneal endothelial cells were evaluated. The mean surface diameter and area of rabbits' central corneal endothelial cells were measured first in vivo by specular microscope. Thereafter the same corneas were fixed in 9 different solutions and processed for scanning electron microscopy. The surface structure of the same endothelium was then photographed with SEM. The cell dimensions were remeasured. According to our results 1.25% or 2.50% glutaraldehydes without sucrose gave the best surface preservation and caused the least shrinkage.

Interface quality of different corneal lamellar–cut depths for femtosecond laser–assisted lamellar anterior keratoplasty

“…Even though the SEM preparation technique used in this study was similar to that used in established studies, 30,31 the dehydration and metal coating of corneas for SEM imaging could introduce shrinkage and microscopic roughness, which might affect the accuracy in qualifying microscopic surface features. 32,33 …”

Ultrastructural morphology and morphometry of the normal corneal endothelium of adult crossbred pig

“…Corneal buttons were obtained by a 360º section 2 to 3mm from the limbic region to the sclera and reduced to 4 portions. Sample preparation for SEM was done following the report of VIRTANEN et al (1984). Samples were transferred to a 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at pH 7.4 and stored for 24h at 4ºC for fixation.…”

Scanning electron microscopy of the corneal endothelium of ostrich

“…Despite of their considerable use, the effects of preparation of cornea for SEM on corneal endothelium have been described (VIRTANEN et al, 1984). SEM, with appropriate consideration of the effects of fixation, allows the evaluation of morphology and morphometric analysis of endothelial cells of ostrich.…”