The keratocyte network of human cornea: A three-dimensional study using confocal laser scanning fluorescence microscopy

Purpose. Keratocytes of the living human cornea were examined to compare qu antitatively spatial arrangement and cell volume of the stromal layers. Thi s knowledge is required for further studies toward a quantitative understan ding of cellular alterations in corneal pathology. Methods. Three human cor neas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) di rectly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of less than or equal to 270 mu m in the z-axis were reconstr ucted. Cell density and volume density were determined by computer-aided mo rphometry. Results. Three keratocyte subpopulations were distinguished. The ir spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (1 00%) to posterior (53.7%) stroma, volume density was highest in the posteri or stroma (17.03 +/- 5.05%) and lowest in the central stroma (9.31 +/- 1.09 %). In the anterior stroma, volume density was found to be 10.19 +/- 4.37%. Conclusion. Confocal laser scanning fluorescence microscopy allowed quanti tative analysis and the visualization of the spatial arrangement of the ker atocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal ce llular arrangements in corneal disease.