Emscf200h cu th

Carbon-coated grids (EMSCF200H-CU-TH, ProSciTech) were glow discharged to render them hydrophilic. A 1–5 μl aliquot of sample was applied to an upturned grid held in anti-capillary forceps, over moist filter paper, and left for 5 min over moistened filter paper to adsorb. Excess liquid was then removed with filter paper, the grid was then inverted onto a drop of 2% PTA stain, pH 6.9 on Parafilm, for 1 min. The grid was removed, the stain wicked away with filter paper and allowed to dry before viewing in the microscope. The samples were examined using Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, Netherlands) at an operating voltage of 120 kV. Images were recorded using a FEI Eagle 4 k × 4 k CCD camera and AnalySIS v3.2 camera control software (Olympus).

Unmodified collagen samples were gelled after neutralising and heating to 37 °C, as with the rheology measurements, however, the TEM samples were diluted (1 mg/mL) so that a weaker gel that could be pipetted onto an imaging grid. Methacrylated samples had LAP added at 0.05%, followed by exposure to 400 nm light at 20 mW/cm² for 30 s prior to taking to 37 °C, following the approach used for the rheology measurements. Gelled samples were examined by transmission electron microscopy (TEM) using a Tecnai 12 transmission electron microscope (FEI, Eindhoven, The Netherlands) at an operating voltage of 120 kV. Samples were prepared on carbon-coated grids (EMSCF200H-CU-TH, ProSciTech, Kirwan, Australia) that were glow-discharged to render them hydrophilic. A 1-microliter drop of sample was applied to an upturned grid held in anti-capillary forceps over moist filter paper, and left for 1 min over moistened filter paper. Excess liquid was then removed with filter paper and the grid was then inverted onto a drop of 2% PTA stain, pH 6.9 on parafilm, for 1 min. The grid was removed from the stain, and excess stain was wicked away with filter paper. Grids were allowed to dry before viewing under the microscope. Images were recorded using a FEI Eagle 4k × 4k CCD camera with AnalySIS v3.2 camera-control software (Olympus, Tokyo, Japan).