Quanta 250 esem

SEM imaging of a mineral-loaded micromotor was obtained with an FEI Quanta 250 ESEM instrument, using an acceleration voltage of 10 kV. EDX mapping analysis was performed using an Oxford EDX detector attached to the SEM instrument and operated by Pathfinder software. For additional characterization, iron(II) gluconate hydrate or sodium selenate decahydrate were loaded in chitosan layers further labeled with FITC (λ_ex=492 nm/ λ_em=520 nm, Sigma-Aldrich, 46945) or Rh-6G (λ_ex=524 nm/ λ_em=547 nm, Sigma-Aldrich, R4127) fluorescent dyes, respectively. Brightfield and fluorescent images of the mineral-loaded motors were captured using an Invitrogen EVOS FL microscope coupled with a 10× and 40× microscope objectives using the GFP and RFP fluorescence filters for green and red excitation, respectively.

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)).

Scanning electron microscopy was performed in the Vanderbilt Cell Imaging Shared Resource. Axial cross-sections of representative acellular nerve samples were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The specimens were post-fixed with 1% OsO4 solution in 0.1 M cacodylate buffer (pH 7.4) for 2 h, dehydrated through a graded ethanol series in PBS, and sputter coated with 3.5 μm of gold. Quanta 250 ESEM (FEI, Hillsboro, OR) was used to capture surface images at 300× to 3000×.