Confocal imaging of the human keratocyte network using the vital dye 5-chloromethylfluorescein diacetate

Background: The human corneal stroma consists of intercalated layers of collagen and keratocytes. These cells are known to maintain the stroma and aid in repair but it is likely they have other crucial roles throughout the cornea. The complexity of their anatomy is revealed in this study by ex vivo in situ images of the human keratocyte covering a range of ages. Methods: Human donor corneas of different ages were stained with 5-chloromethylfluorescein diacetate (CMFDA), a dye that is anchored and retained within the cell cytoplasm. The tissue was fixed, sectioned, mounted, and then imaged using a confocal laser scanning microscope at various magnifications and tissue planes. The digital image sets were transferred to multifunction image processing software for analysis and production of 3-D stereo images of keratocyte networks throughout the stroma. Results: High quality images of CMFDA-stained cells revealed differences in the structure and orientation of keratocytes in the anterior, central and posterior stroma, which did not differ throughout the age-range studied. This method reveals very fine cell process ramifications not previously visualized, orientated in lateral and antero-posterior directions, and it confirms the potential for multidirectional communication between keratocyte networks. Conclusions: This qualitative study found consistency of keratocyte morphology in the normal human cornea throughout life. It confirmed differences in keratocyte anatomy, and the potential for rapid cellular communication by multiple interconnecting processes supporting cohesive keratocyte activity. This high-resolution 3-D microscopic study should assist in identifying gross deviant cellular behaviour in post-surgical and disease states.