Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization

Abstract

Collagen fibrils are the major tensile element in vertebrate tissues where they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess formation of the fibrils, predominantly by measuring fibril diameter. Here we describe an enhanced protocol for measuring fibril diameter as well as fibril-volume-fraction, mean fibril length, fibril cross-sectional shape, and fibril 3D organization that are also major determinants of tissue function. Serial section TEM (ssTEM) has been used to visualize fibril 3D-organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View®). We demonstrate the power of 3View® for studying collagen fibril organization in vivo and show how to find and track individual fibrils.Time scale: ~8 days from isolating the tissue to having a 3D image stack.

3‐D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling

“…Biopsy samples were prepared for electron microscopy as described previously (Starborg et al, 2013). In brief, 1×1×1 mm cubes of tendon were immersed in 2.5 % glutaraldehyde prepared in 100 mM cacodylate buffer (pH 7.2), and processed using a double osmium protocol that is suitable for transmission electron microscopy and scanning block face-scanning electron microscopy (Starborg et al, 2013).…”

“…Ultrathin sections (70 nm-thick) were prepared and fibril diameter measurements were made using a FEI BioTwin transmission electron microscope. Resin blocks were trimmed and images were collected using an FEI Quanta 250 ESEM equipped with a Gatan 3View® for in-chamber ultramicrotome sectioning and image acquisition, as described previously (Starborg et al, 2013). Typically, 500–1000 × 100 nm-thick cuts were removed from the blocks during the imaging procedure and image analysis and model reconstruction was performed using IMOD (Kremer et al, 1996).…”

“…However, difficulties in obtaining undistorted sections and in series have precluded a detailed ultrastructural study of tissue organization in tendinopathy. Recent application of serial block face-scanning electron microscopy (SBF-SEM) to studies of embryonic tendon tissue has shown that this technique is particularly useful for studying long-range collagen fibril organization (Starborg et al, 2013). …”

Techniques to assess bone ultrastructure organization: orientation and arrangement of mineralized collagen fibrils

“…It should be noted that both techniques also allow investigations of bone microporosities, including the lacuno-canalicular network [245]. Still, the extended sample preparation protocols and the extended imaging times involved for serial sectioning electron microscopy consume a lot of resources and time [246].…”

“…The result is stacks of 2D images that provide a 3D volume of the sample. Given that the result of such a procedure is a micrometre volume with nanometre resolution, the data can be used to study ultrastructure organization of tissues such as muscle, tendon [207] or bone, either in two dimensions [208,244,245] or in three dimensions [9,246] ( figure 14). Although only a limited tissue volume can be assessed when using volume electron techniques (the spatial resolution at nanometre levels restricts the FOV to tens of micrometres), and despite the fact that these techniques are destructive, they provide important insights concerning the organization of the bone ultrastructure, such as in Haversian systems [9] or trabeculae [247].…”