A comparison of fixation methods for SEM analysis of self-assembling peptide hydrogel nanoarchitecture

Determining the porosity of hydrogels is an important component of material characterisation. While scanning electron microscopy (SEM) is a widely used method to study hydrogel nanoarchitecture, it is well-established that SEM sample preparation methods can alter the structure of hydrogels. Herein we describe the impact of sample preparation on the SEM analysis of self-assembling β-peptide hydrogels. Three methods of hydrogel preparation for SEM were compared, and each method preserved distinctly different nanoarchitecture, specifically, different levels of fibre alignment and porosity. Comparison of conventional SEM preparation and our hybrid method, which comprises high pressure freezing, freeze substitution without fixative and critical point drying, showed a high degree of similarity at the nanometre scale and diverging architecture at the micron scale. This study quantified the impact of chemical fixation versus high pressure freezing on self-assembling β3-peptide hydrogels, demonstrated the effect of sample preparation on fibre alignment and porosity, and presents a novel hybrid preparation method where chemical fixation can be avoided when conventional SEM is desired.