Scanning electron microscopy operated at cryogenic temperature (cryo-SEM) is a powerful tool for investigating surface and cross-sectional nanostructures of water-containing samples. Typically, cryo-SEM samples are frozen just before observation in specific metal carriers. However, pre-frozen samples are also of interest, such as frozen food and freeze-stored animal samples. In such cases, sample mounting with a defined orientation is required, but there has been a lack of ideal conductive adhesives that can be used without increasing the sample temperature. Here, we developed a mixture of graphite oxide and 1,3-butanediol as an adhesive, capable of gluing samples at dry-ice temperature and is frozen below that temperature. Dispersion of graphite oxide increased the conductivity and reduced the charge-up contrast. Acquisition of energy-dispersive X-ray spectrum, cross-sectional ion milling, and high-resolution imaging were successfully achieved using the adhesive. We tested and confirmed the usefulness of this new adhesive by applying it to cryo-SEM surface imaging of diatomite, freeze-fractured cross-sectional imaging of chicken liver, and ion milling cross-sectional imaging of a deep-sea snail. The new adhesive is not only useful for food science and field-preserved biological samples but also potentially applicable to wider fields such as archaeological and biological samples preserved under permafrost.
Keywords: freeze-fracture; graphite oxide; low-temperature imaging; scanning electron microscopy; specimen handling.