A Cryo-/Liquid Phase Correlative Light Electron Microscopy Workflow to Visualize Crystallization Processes in Graphene Liquid Cells

Liquid phase electron microscopy has emerged as a powerful technique forin-situobservation of material formation in liquid. However, the interaction of electrons with an aqueous environment leads to the decomposition of water molecules into multiple radicals (radiolysis), which can affect the formation process. Graphene’s ultra-conductive properties have made it an efficient way to mitigate this problem, as it acts as an electron scavenger when used as a window material. However, finding accessible and reliable protocols for the formation of graphene liquid cell (GLC) has been an enormous challenge. Even though loop-assisted transfer has recently been demonstrated as an effective method, the liquid cells are randomly formed and the process of interest is initiated when the GLCs are sealed. This means that the process cannot be imaged at early timepoints, because searching for their location is a time-consuming effort during which the sample is exposed to electrons.Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses some of the limitations of the graphene liquid cells, combining the advantages of fluorescence and electron microscopy. Fluorescence microscopy (FM) is used to locate the GLCs without exposure to electrons and electron microscopy to capture dynamics with nanoscale resolution. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at the moment of interest. We demonstrate the workflow by observing non-classical crystallization pathways, but also highlight its potential application in biological systems.