Abstract
Purpose: :
Corneal transparency relies on the constant renewal of epithelial cells in which stem cells are believed to play a crucial role. Despite recent advances in stem cell biology, it is not yet possible to identify individual stem cells in the ocular surface in situ. As an initial step toward testing a hypothesis that stem cells could be identified by their spectral properties, we examined whether individual ocular surface epithelial cells could be distinguished from each other based on their spectral profile.
Methods: :
Whole-mount ocular surface preparations of C57BL/6J and A/J mice were stained with a nuclear fluorescent dye, Hoechst 33258. Nuclear heterochromatin foci were characterized and fluorescence emission spectra were obtained from individual basal epithelial cells of the cornea, limbus and conjunctiva under a fluorescence microscope fitted with a liquid crystal tunable filter. As a measure of epithelial differentiation, some specimens were analyzed for a telomere length by hybridization with a PNA probe. In the limbus, individual cells were analyzed for the correlation among the heterochromatin foci, the spectral profile, the telomere length, and the location expressed as a distance from a cornea-limbus boundary line.
Results: :
Limbal basal epithelial cells above the limbal capillary arcade contained heterochromatin foci that could be clearly distinguished from those of the cornea or the conjunctiva. As a group, these cells exhibited a different spectral pattern from that of the cornea and the bulbar conjunctiva, and they contained the longest telomere in the ocular surface as reported previously by Blasco's group. Within the limbus, a variation of the spectral pattern among different cells was small but significant, allowing discrimination of these cells. Combined with heterochromatin foci, telomere length, and the location, each limbal epithelial cell could be uniquely identified.
Conclusions: :
Each ocular surface epithelial cell exhibits a unique Hoechst 33258 emission spectrum. This spectral property allows discrimination of: 1) corneal cells, limbal cells, and conjunctival cells, 2) individual cells within the cornea, the limbus or the conjunctiva. Spectral differences may reflect the state of epithelial differentiation, and if so, spectral imaging would be a useful adjunct to existing techniques for identifying stem cells in situ.
Keywords: cornea: epithelium • microscopy: light/fluorescence/immunohistochemistry • imaging/image analysis: non-clinical