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S. X. Deng, K. Mohd Kamal; Study of Three-Dimensional Architecture of Human Limbus in Whole- Mount Tissue by Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6068.
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© ARVO (1962-2015); The Authors (2016-present)
To visualize the three dimensional (3-D) structure of the human limbal epithelium stem cell (LESC) niche using confocal microscopy
Transplant graded corneo-scleral rims were fixed in 4% paraformaldehyde. The cell nucleus were labelled with Hoechst 33342 or Propodium Iodine (PI), and the cytoplasma with Alexa 488 conjugated Phalloidin. Basal limbal epithelial cells were identified by immunohistochemistry technique using anti-P63 antibodies. Sequential optical sections of the limbal area in the whole-mount tissue confocal microscopy images were acquired using a Leica TCS-SP confocal microscope. Images were analysed and the 3-D structure were reconstructed using the Leica Confocal. Software.
The optimal condition to label and visualize the limbal epithelium and the underlying stromal cells were determined. Reconstructed 3-D architecture of the limbus revealed limbal crypts (LP) and focal stromal projections (FSP) structures. Double labelling experiments with PI and the putative LESC marker P63α using immunohistochemistry technique demonstrated the location of P63α + cells in a 3-D configuration. The organization of blood vessels within the limbal region was also characterized.
This study confirms the existence of limbal crypts and FSP within the limbal region and is the first to visualize the spatial orientation of the P63α + potential LESCs and their niche in wholemount tissue. This new technique could be used to further characterize the LESC niche.
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