Novel Fine Bamboo Pillar Assisted Freeze-Fracture Method for Hydrogel Sample Preparation of Cryogenic Scanning Electron Microscope

Cryogenic scanning electron microscopy (cryo-SEM) is an irreplaceable approach to investigate the real surface morphology and microstructure of hydrated samples. The freeze-fracture of samples is one of the most important pretreatments to obtain ideal results. Due to the high content of water, hydrogel samples are very soft and difficult to handle when performing cryo-SEM sample preparation. During the freeze-fracture procedure using conventional fracture method with cold knife, it is difficult to obtain high-quality fractured surface and the efficiency is rather low. Therefore, a novel method of the freeze-fracture for hydrogel samples by employing fine bamboo pillars for assistance has been proposed. Before the cryo-fixation of samples, fine bamboo pillars were inserted at the top of the hydrogel samples. During freeze-fracture process, the cold knife would pry the fine bamboo pillars away and make a cleavage of hydrogel sample instead of hitting the sample directly to achieve freeze-fracture in the area of contact between the sample and the fine bamboo pillar. By the method, a large area of fractured surface could be revealed for SEM investigation. The damage to sample caused by stress was reduced, and a clear sample surface free of debris was provided. Meanwhile, multiple samples were fractured simultaneously at one time, which significantly enhanced the efficiency of freeze-fracture.