Abstract
Eye banked research-grade human donor corneas serve as principal ex vivo source for studying the mechanisms that underlie corneal endothelial cell damage/death and survival. Wide-field specular microscopy can be used for corneal endothelial visualization and allows for indirect assessment of endothelial cell function by analyzing endothelial cell density and morphometric parameters. However, a standardized approach is needed to observe corneal endothelial changes over time. This protocol describes reliable ex vivo methods for consecutive analyses of human donor corneal endothelial cell density and morphometric parameters change using a wide-field dual imaging specular microscope. This protocol involves tissue warming, acquisition and analysis of specular endothelial images, assessment of corneal layers with the new Enhance mode, optical pachymetry measurement, and qualitative image quality grading scales. This quantitative and qualitative evaluation of donor corneas allows for a systematic analysis of endothelial dynamic responses to ex vivo induced stress and can be used as a valuable tool to better elucidate specular findings and mechanisms mediating corneal endothelial cell loss in corneal disease and after transplantation.