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

Material formation and development in liquid is an important phenomenon in both synthetic and biological environments. To better understand the mechanisms of material formation, we must visualize the dynamics of the process in combination with (bio)chemical information. Although cryogenic transmission electron microcopy (cryo-TEM) is commonly used to elucidate mechanisms of material development in hydrated conditions [1, 2], it still does not provide dynamic information. Developments in liquid phase electron microscopy (LP-EM) over the last decades have made it possible to combine in-situ observation of material formation with high-resolution chemical information [3, 4]. However, the interaction of electrons with a liquid layer generates a complex sequence of chemical reactions (radiolysis) resulting in the formation of intermediate species that influence the reaction. This makes the dose tolerance for both synthetic and biological materials even lower than for other electron microscopy techniques [3, 5]. The use of graphene as encapsulating material has become a way to reduce this problem. Due to its ability to mitigate reactive species [6], graphene is able to reduce electron damage, thereby increasing the critical electron dose. In addition, its ultrathin structure and electrotransparency significantly increase the resolution compared to standard SiN windows [7, 8]. However finding accessible and reliable protocols for graphene liquid cell (GLC) formation has been an enormous challenge [9]. Even though loop-assisted transfer (LAT) has recently been demonstrated as an effective method for GLC preparation [10], graphene remains fragile during manual handling. As GLCs are closed volumes, the process of interest will be initiated the moment the GLCs are sealed. This means that process cannot be imaged at early timepoints, in particular because GLCs are formed randomly on the grid and searching for their location is a time-consuming effort. Furthermore, during the searching process in the TEM the sample will be exposed to the electron beam, which will decrease the number of electrons available for imaging. We present a novel workflow which combines the benefits of correlative cryogenic light electron microscopy (cryo-CLEM) [11] with LP-EM (Fig. 1). The workflow consists of two main parts: 1. the graphene liquid cell preparation and 2. the correlative imaging sequence. First, the sample is encapsulated in graphene using automated loop-assisted transfer [12], where touching the grid with the liquid in the loop will start the reaction. Thereafter, the grid is vitrified at the start of the process of interest. This can be either immediately after encapsulation, or after using live-light microscopy (live-LM) to determine an interesting starting point. Vitrifying the grid will arrest all processes in a cryogenic state. This allows for the localization of the region of interest by cryo-LM without the continuing of the process. Subsequently, the starting point for the investigation is imaged in high resolution with cryo-TEM. The grid is then thawed inside the microscope for capturing the dynamics of the revived process with LP-TEM. To confirm the presence of liquid after thawing, the grid is imaged with live-LM. To demonstrate the workflow, a crystallization buffer together with a non-reactive fluorescent dye was encapsulated in graphene using an automated method [12], which results in higher encapsulation efficiency, and vitrified to arrest the crystallization process (Fig. 2a). Cryo-LM was used to locate the liquid pockets on the vitrified grid (Fig. 2a). Overlay of LP-TEM overview with cryo-FM was used to select ROIs for high resolution imaging (Fig. 2b). The grid was then heated-up to room temperature inside the microscope to re-start the reaction (2c-d). Different mechanisms of crystal growth such as particle attachment, nanoparticle aggregation, dissolution/reprecipitation, amorphous-to-crystalline phase transformation and Ostwald ripening were observed. In summary, the cryo-/LP-CLEM workflow presented here allows for the observation of material formation processes at a predetermined time and space. The workflow combines the benefits of both cryo-TEM and LP-TEM and could have a significant impact on the visualization of dynamic biological processes [13]. Schematic representation of the cryo-/LP-CLEM workflow indicating the imaging sequence including the temperatures. Correlative cryo-/liquid phase imaging of graphene liquid cells. a) Overview of the grid after vitrification showing the GLCs. b) Overview of the grid after thawing. Fluorescent microscopy (green) is used to indicate the GLCs. c) Higher magnification of the red box showing multiple small crystals inside a GLC. d) Coalescence of the crystals inside the GLC.