Pp3010

FIB-SEM imaging was performed using FEI Helios NanoLab 660 (Thermo Fisher Scientific) equipped with a cryo-preparation system (PP3010, Quorum). The sample preparation procedures were as follows: hydrogel samples were rinsed with PBS and then fixed with 4% glutaraldehyde at 4 °C overnight and then post-fixed with 1% OsO₄ in ddH₂O for 1 h. The samples were further washed with PBS three times. The hydrated samples were carefully mounted and quickly frozen by dipping them into liquid nitrogen. The frozen samples were transferred onto a cryo-stage (−140 °C) and fractured with a knife in the Quorum chamber. Thereafter, the temperature of the stage was set to −85 °C and then gradually increased to −50 °C (5 °C/min). The temperature was maintained at −50 °C for five more minutes, after which it was reduced to −140 °C. The sublimed samples were sputter-coated with platinum in the SEM (10 mA, 60 s) to increase the conductivity. The images were acquired with a 50-pA beam current at a 5-kV acceleration voltage using the concentric backscatter detector of the SEM.
To calculate the average pore sizes and number densities of hydrogels in the SEM images, we used the Analyze Particle function in ImageJ. For each type of hydrogel, we independently prepared two samples and acquired four SEM images on each sample. The field of view of each image was 31.5 × 22.5 μm².

To obtain the SEM images, Aqua Sperm micromotors were fixed for 30 min in 1% glutaraldehyde at RT, washed with PBS, and then centrifuged at 600×g for 10 min. Aqua Sperm were dehydrated for 5 min in 40%, 50%, 60%, 70%, 80%, 90%, and for 10 min 100% ethanol; the final 100% alcohol was replaced twice. Final fixation was in hexamethyldisilazane for 30 min. SEM images were obtained using a scanning electron microscope, Tescan MAIA 3 (Tescan Ltd., Brno, Czech Republic) equipped with a field emission gun, cryogenic system PP3010 (Quorum Technologies Ltd., Sussex,UK).

The microstructures of WPF under different pH values were characterized using atomic force microscopy (AFM, Bruker, Germany). First, 2% (w/v) solutions were diluted 400 times to get a final concentration of 0.005% (w/v) fibril, sequentially 20 μL dispersion was dropped onto freshly prepared cleaved mica and equilibrate for 3 min. Then, the redundant samples were removed by rinsing with deionized water and dried under flow nitrogen. AFM tests were conducted with a Dimension edge (Bruker, Germany) using tapping mode at ambient temperature. The microstructures of WPF-GNP hydrogel and WPI-GNP hybrid (4:1, pH 4.0, heat for 3 h) were further detected by Cryo-scanning electron microscope (Cryo-SEM, SU8000, HITACHI); first, the samples were placed on a mental deck and then put into liquid nitrogen for a few seconds to solidify the samples. Second, the frozen samples were cut by a microtome (PP3010, Quorum Technologies Laughton, England) to obtain a flat surface and then do a gold sputtering. Third, the mental deck was transferred to a distillation device to sublimate the water (–95°C, 20 min). Finally, the microstructures of the samples were characterized.