CONFOCAL LASER SCANNING MICROSCOPIC EXAMINATION OF TRANSPORT PATHWAYS AND BARRIERS OF SOME PEPTIDES ACROSS THE CORNEA

Recent advances in optical microscopy involving confocal imaging are now becoming available which dramatically improve resolution, contrast, and rejection of out-of-focus noise as compared to conventional imaging. With this technique, it is possible to optically section thick specimens, allowing interior structures of living specimens that are normally obscured in conventional imaging to be visualized. The unique transparency of the cornea makes microscopic examination by this technique particularly applicable. In the present study, the technique, performed in both light-reflected and fluorescence modes, was used to obtain information about cellular structures, transport pathways and barriers of two peptides, poly-l-lysine and insulin in the cornea. In the light-reflected mode, serial images showing detailed structures of various layers of the cornea including the epithelial surface, wing cells, basal cells, stroma, and endothelium were clearly demonstrated. The transport pathways of fluorescently labeled peptides were studied using the fluorescence mode. The results suggest preferential uptake of polylysine through epithelial surface defects caused by non-uniform shedding of superficial 'old' cells and through intercellular spaces of the cornea. In contrast, corneal uptake of insulin was found to occur predominantly via intemalization of the peptide by the surface cells. In both cases, the penetration of these peptides was severely limited to the outermost layer of the corneal epithelium. The results also indicate charge discrimination effects to penetration of negatively charged insulin by the cornea. © 1990.