A Cryo-to-Liquid Phase Correlative Light Electron Microscopy Workflow for the Visualization of Biological Processes in Graphene Liquid Cells

Liquid phase electron microscopy (LP-EM) has emerged as a powerful technique for in-situ observation of material formation in liquid. However, monitoring these processes requires the repeated interaction of the electron beam with the aqueous environment leading to the decomposition of water molecules (radiolysis), which affects the formation processes under investigation. Graphenes 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. Using the strategy, the process of interest is initiated when the graphene liquid cells (GLCs) are sealed. This means that the process cannot be imaged at early time points since microscope preparation and initiation of image acquisition at the region of interest are time-consuming. Here we report a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses the most significant limitations of the graphene liquid cells, while combining the advantages of fluorescence and electron microscopy. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at a selected time point. We demonstrate the workflow first by observing multiple day crystallization processes and highlight its potential by observing a biological process: the complexation of calciprotein particles. With this observation, we show the exciting possibilities for LP-EM in biology.