The micropore conundrum – examining the preparation of hydrogels for ultrastructural analysis

Large numbers of papers which include studies on hydrogels use SEM images to analyze pore sizes, often demonstrating so-called micropores of few to tens of micrometers in size. Commonly, hydrogels are lyophilized, which often involves freezing under insufficiently controlled conditions. Considering experience from cryo-electron microscopy and the behavior of water during freezing, the obvious question arises: Was the freezing process fast enough to avoid formation of ice crystals? If not, freeze drying will lead to phase separation and formation of voids and pores. Here we show, using pNIPAM hydrogels as example, that freezing can indeed produce micropores. We propose an alternative, chemical preparation for hydrogels based on infiltration with tannic acid allowing ultrastructural analysis without creating artificial micropores. To study the hydrogel samples’ interior, they were either fractured or embedded into an epoxide resin for sectioning by ultramicrotomy. Analysis of hydrogels prepared in a controlled, chemical way revealed no micropores, but pores in the range of 20-100 nanometers for a pNIPAM formulation with high water content. Two-photon printed microstructures with intrinsically lower water content could also be stabilized by the chemical protocol and sections showed corresponding nanopores. Preparation with tannic acid thus provides a convenient route to fast and artefact-minimized screening and benchmarking of structured hydrogels.