Eagle 4 k 4 k ccd camera

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).

A laboratory-built humidity-controlled vitrification system was used to prepare the samples for Cryo-TEM. Humidity was kept at 80% for all experiments, and ambient temperature was 22°C. 300-mesh copper grids coated with perforated carbon film (Lacey carbon film: ProSciTech GSCU300FL-50, Kirwan, Qld, Australia) were glow discharged using a Pelco EasiGlow Glow Discharge unit to render the carbon film hydrophilic. 3 μl aliquots of the sample were pipetted onto each grid prior to plunging. After 5 s adsorption time the grid was blotted manually using Whatman 541 filter paper, for 4 s. The grid was then plunged into liquid ethane cooled by liquid nitrogen. Frozen grids were stored in liquid nitrogen until required. The samples were examined using a Gatan 626 cryoholder (Gatan, Pleasanton, CA, USA) and Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, Netherlands) at an operating voltage of 120 kV. At all times low dose procedures were followed, using an electron dose of less than 5 electrons/Ų for all imaging. Images were recorded using a FEI Eagle 4 k × 4 k CCD camera at a range of magnifications using AnalySIS v3.2 camera control software (Olympus).