SEM Sample Preparation Protocol

For SEM analysis, samples were processed following previously reported protocols [32 (link)]. After 8 h of incubation, the samples were fixed overnight at 4 °C in a 4% paraformaldehyde (TAAB, Berks, UK) and 2.5% glutaraldehyde (TAAB, Berks, UK) in 0.1 M phosphate buffer (ph = 7) (Thermofisher, Waltham, MS, USA). Sample fixation was progressed by three sequential steps at room temperature involving aqueous solutions of 2% osmium tetroxide (TAAB, Berks, UK) for 1 h, 1% tannic acid (TAAB, Berks, UK) for 30 min and 2% osmium tetroxide for 1 h, followed by overnight incubation in 1% uranyl acetate solution in water at 4 °C. Abundant rinsing with DI water was performed between each fixation step. The samples were then rinsed with DI water and progressively dehydrated using increasing ethanol concentrations (i.e., 30%, 50%, 70%, 90% and 100%), CO₂-critical-point dried (Quorum Technologies K850, Sacramento, CA, USA) and subsequently sputter coated with 10 nm of Au/Pd (Quorum Technologies Q150T, Sacramento, CA, USA) for SEM imaging at 10 kV using a JEOL7001F FE-SEM system (Jeol LTD, Akishima, Japan).