Basic properties of solidified organic liquids at a cryogenic temperature for electron microscopic visualization and sample preparation of dispersion systems

It is challenging to image structures in liquids for electron microscopy (EM); thus, low-temperature imaging has been developed, initially for aqueous systems. Organic liquids (OLs) are widely used as dispersants, although their cryogenic EM (cryo-EM) imaging is less common than that of aqueous systems. This is because the basic properties (e.g. vapor pressure, density and amorphousness) of OL in the solid state have not been extensively investigated, preventing the determination of whether the observed structure is free from artifacts. Herein, I summarized physical data related to the phase change, and the solid density at 77 K and sublimation speed for some OLs were measured independently to discuss the applicability of OLs for cryo-EM. Among various OL properties, the sublimation temperature, pressure and rate and crystallinity are important for cryo-EM. The sublimation-related properties are used to judge whether the OL is stable during storage, observation and sample preparation such as etching. These properties were calculated, and the calculated sublimation speed matched with that measured by cryogenic scanning EM movie imaging. Crystallinity was estimated using the difference between the extrapolated temperature-dependent liquid density and the solid density of frozen OLs measured in liquid nitrogen. Artifacts observed upon freezing were exemplified by focused ion beam cross-sections of OL-in-water emulsions, and cracks, voids and wrinkles are found in the OL phase at a large shrinkage ratio. The study findings show that the applicability of OLs largely differs for structural isomers and that appropriate OLs are required for the cryo-EM imaging of nonaqueous systems.