Fluorescence microscopy and three-dimensional imaging of the porcine corneal keratocyte network

Background: Little is known about the spatial arrangement and the corresponding morphometric data describing the living keratocyte network. For determination of alterations in corneal diseases it is crucial to know the morphology of the keratocyte network in the healthy state. Porcine cornea was used as a model tissue because it allows the study of species differences. Methods: Corneas from freshly enucleated pig eyes were stained with calcein ANI and ethidium homodimer and examined by confocal laser scanning microscopy. High-resolution fluorescence images were used for three-dimensional reconstructions from which cell density and volume density were determined by computer-aided morphometry. Results: Three keratocyte subpopulations were distinguished and visualized in their spatial arrangement. Significant differences with respect to both shape and fluorescence intensity distribution of the cell bodies were found. Cell volume density was 7.7% in the anterior stroma, 13.7% in the central stroma and 11.8% in the posterior stroma. Conclusion: The technique described allowed good visualization of the spatial arrangement of the keratocyte network. Combined with morphometric methods, the analysis of the state of the cornea yields a quantitative description. The method is expected to be useful for the determination of morphological alterations in corneal disease or following surgical treatment.