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C. Pinali, R. D. Young, A. J. Quantock, P. N. Lewis, C. Knupp; Electron Tomography Reveals the Three Dimensional Arrangement of Proteoglycans and Collagen in High Pressure Frozen Mouse Corneas. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6206.
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© ARVO (1962-2015); The Authors (2016-present)
Collagen and proteoglycans define the structure and the function of cornea. To evaluate the way in which they interact in three dimensions, we cryofixed some murine corneas (some with and some without pretreatment with the cationic dye cuprolinic blue (CB) ) before carrying out a three dimensional tomographic reconstruction.
Corneas were obtained from wild type mice. They were prepared in two ways: 1) After excision corneas were high pressure frozen using a Bal-Tec HPM 010 and stored in liquid nitrogen. They were freeze substituted at -90°C with 1% osmium tetra-oxide dissolved in anhydrous acetone and embedded in epoxy resin at room temperature. 2) Freshly excised corneas were stored in 4% paraformaldehyde before fixation in 2.5% glutaraldehyde in 25 mM sodium acetate buffer with 0.05% CB and 0.1 M MgCl2. Cryofixation was achieved with a Leica EMPACT2. After freeze substitution in acetone, resin embedding was performed at -50°C with Lowicryl HM20 resin. Sections from both specimens were examined in a JEOL 1010 transmission electron microscope at 80 KV and tilt series were collected between 60° and -60°. Alignment of tilt series and three dimensional reconstructions were made with IMOD and EM3D.
The overall three dimensional appearance of proteoglycans and collagen in all specimens examined was very similar. Proteoglycans formed bridges between the collagen fibrils with no apparent rotational symmetry about the fibrils, but an extensive network of proteoglycans was seen that connected several neighbouring fibrils. Their appearance was also essentially identical to previous three dimensional reconstructions from mouse corneas carried out by treating the specimens with conventional, room temperature methods (see poster by Parfitt et al.) indicating that CB does not create visible artefacts at the resolution obtained in these studies.
Electron tomography provides a clear picture of the collagen proteoglycan arrangement in the cornea. The ordered arrangement of the collagen fibrils in the cornea is obtained through the action of the proteoglycans that form an extensive network between the fibrils. The use of CB during the preparation of the specimens does not seem to disrupt this arrangement.
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