Cryo-FIB-SEM visualization and radiation damage of a water–THF phase-separated mixture and <i>in situ</i> formed organic nanoparticles

Cryoelectron microscopy is a powerful technique for high-resolution imaging of nonaqueous liquids, but challenges remain regarding imaging and data interpretation. Recent advancements in estimating the physicochemical properties of pure organic liquids at cryogenic temperatures have enhanced the selection of imaging and pre-treatment conditions. However, whether binary mixtures behave similarly to pure substances is still unclear. Furthermore, focused ion beam (FIB) milling facilitates site-specific cross-sectioning, but its effects on the microscopic morphology of frozen organic liquids are not well understood. In this study, we investigated water-tetrahydrofuran (THF) binary mixtures as a model to explore their phase behavior and radiation damage under cryogenic conditions. Spectroscopic analyses indicated microscopic phase separation within the seemingly miscible water-THF mixtures, but their detailed structure has been a subject of ongoing debate. Using cryo-scanning electron microscopy with FIB (cryo-FIB-SEM), we visualized bicontinuous phase-separation. The domain sizes were consistent across spectroscopic data, thermally sublimed surfaces, and FIB cross-sections. Notably, FIB milling caused a significant loss of THF-rich regions, likely due to localized temperature increases of approximately 178 K, which is an order of magnitude greater than that in water-rich domains. We also noted the nanoparticles of electron-resistant carbazole-terminated carbon-bridged oligo(para-phenylenevinylene) (COPV2-G1) formed within the THF-rich phase. Extended electron irradiation led to morphological changes and shrinkage, suggesting THF was incorporated into COPV2-G1 aggregates along with THF decomposition induced by the electron beam. These findings underscore critical considerations in cryo-FIB-SEM imaging of binary organic liquids and solvated particles, providing practical insights for reducing or leveraging ion/electron beam-induced artifacts.