Biotwin transmission electron microscope

Biopsy samples were prepared for electron microscopy as described previously (Starborg et al., 2013 (link)). 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 (link)). Semi-thin (~1 μm thick) sections were prepared, stained with toluidine blue and examined using a dissecting microscope to determine the orientation of the tendon within the biopsies. The resin blocks were trimmed for transverse sectioning (i.e. 90° to the tendon long axis). 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 (link)). 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 (link)).