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Robert D. Young, Christian Pinali, Kenneth M. Png, Andrew J. Bushby, Carlo Knupp, Andrew J. Quantock; Observations on the Formation of Stromal Lamellae by Keratocytes in Developing Cornea using Volume Electron Microscopy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3429.
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© ARVO (1962-2015); The Authors (2016-present)
Mechanisms by which keratocytes synthesise the exquisitely-ordered, lamellar matrix of the transparent cornea remain incompletely understood. To gain a better understanding of the sequence of lamella formation we examined embryonic avian corneas using Volume Electron Microscopy to image stromal development in three dimensions.
Corneas from developing chicks at embryonic day(E)10, E14 and E18 were aldehyde-fixed, postfixed in 1% osmium ferricyanide, then 1% tannic acid to enhance contrast and conductivity, and embedded in Durcupan epoxy resin. En face, semithin toluidine blue-stained sections and unstained ultrathin sections for light and transmission electron microscopy, respectively, aided location of suitable sites for three-dimensional imaging. Selected areas were then milled in the polished resin block faces and imaged using a BSE detector in a FEI Quanta 3D FEG FIB/SEM via an alternate slice-and-view protocol. Milling and 50nm slicing were performed with a focused gallium ion beam, and up to 640 images were collected over 18 h. Image stacks were analysed using ImageJ software.
Three-dimensional reconstructions derived from raw image stacks of stromal volumes up to 4.5 x 104 µm3 showed complex ramifications of keratocytes, with filopodia extending more than 50 µm into the extracellular space. Orthogonal arrangement of cells was evident at E10. At E10, E14 and E18, cells occupied approximately 18, 20 and 20%, and lamellae approximately 20, 50 and 70% of stromal volume, respectively. Interlamellar spaces reduced markedly between E10 and E18. Filopodia were aligned with collagen bundles at E10, appeared central to collagen accumulation in lamellogenesis through E14, and became less evident with stromal condensation and flattening of lamellae at E18.
Volume Electron Microscopy revealed the complexity of cellular morphology in developing chick cornea where prospective keratocytes express extensive filopodial processes which maintain close associations with collagen fibril bundles that increase in size and flatten over the E10 to E18 timeframe. Filopodia may represent a dynamic system through which cells exert control over the spatial and directional organisation of newly synthesised collagen into an emergent lamellar architecture.
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