Abstract
The sclera as well as the cornea forms the principal part of the outer fibrous coat of the eye, with a primary function of protecting the intraocular contents and maintaining the shape of the globe. However, the exact morphometric arrangement of scleral fibroblasts remains unclarified. The aim of this study was to observe the three-dimensional structure of the mouse scleral fibroblasts by focused ion beam/scanning electron microscopy (FIB/SEM). Four eyes from C57BL/6J mice were fixed using a mixture of glutaraldehyde and formaldehyde. The sclera was cut out at the equatorial portion and the posterior pole, and postfixed with potassium ferrocyanide, osmium, thiocarbohydrazide, uranyl acetate and lead aspartate. Specimens were then dehydrated and embedded in an epoxy resin. Serial block face images were obtained using FIB/SEM. Three-dimensional image reconstruction and segmentation of the image stack were created using computer software (Amira v6.0.1, FEI). Scleral fibroblasts were arranged in collagenous layers. The cells frequently showed a cellular junction with the neighboring cells and formed cellular networks. Compared with equatorial fibroblasts, there was a more complicated cellular arrangement of the posterior scleral fibroblasts.
Highlights
The corneal stroma and the sclera form the principal part of the outer fibrous coat of the eye, with a primary function of protecting the intraocular contents and maintaining the shape of the globe
The scleral fibroblasts were located among multiple collagenous layers and appear to have a spindle shape with thin and fine processes (Fig. 1A,B)
We used focused ion beam/scanning electron microscopy (FIB/scanning electron microscopy (SEM)) to examine the three-dimensional ultrastructure of the scleral fibroblasts in mice
Summary
The corneal stroma and the sclera form the principal part of the outer fibrous coat of the eye, with a primary function of protecting the intraocular contents and maintaining the shape of the globe. The corneal stroma is a dense connective tissue consisting of regularly arranged collagenous lamellae and fibroblasts known as keratocytes[1,2,3]. Keratocytes connect to their neighboring cells by gap junctions with fine cell processes and form cellular networks[4,5,6]. An alternative to the serial section TEM approach for imaging tissue has been described for scanning electron microscopy (SEM). The milled face is imaged using the scanning electron beam Since this technique can be highly automated, this removes many of the problems associated with the manual serial section manipulation and imaging in TEM. The aim of this study was to clarify the cellular arrangement in scleral fibroblasts
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