High-quality Scanning Electron Microscopy Incorporated with a Freeze-drying and Gaseous Nitrogen-based Approach for Cell-extracellular Vesicle Interactions

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Scanning electron microscopy (SEM) has offer unique capability for viewing the fine ultrastructure associated with external cellular boundaries, while water-rich (∼80% by volume) cells surrounded by a lipid-rich membrane present a challenge in achieving optimal preservation of topography at high resolution due to the requirement for dehydration and maintenance of membrane fatty acids without compromising membrane properties. Early days of SEM protocols using ethanol resulted in suboptimal preservation of membranes [1, 2]. Application of osmium tetra-oxide (OsO4) [3] and lowering the temperature of reagents [4] medicate the viewing cytoarchitecture. We developed a new sample preparation method for SEM by omitting organic solvent and OsO4 treatments to aim at preserving native molecular organization and enhancing its viewing capability of cytoarchitecture by investigating a freeze-drying method with gaseous nitrogen, FDGN and we demonstrate that samples prepared by this method exceedingly detailed topography via SEM. To test the capabilities of FDGN, we modeled dynamic pathophysiological events, interactions between cultured human cells and tumor cell-derived extracellular vesicles (TEVs). We applied FDGN to investigate changes in human lung fibroblasts (WI-38 cells), as this cell line takes up TEVs shed by breast cancer cells and subsequent TEV uptake is linked to cancer metastasis [6]. The capability of FDGN to preserve detailed structures and retain fluorescent microscopy (FLM) signals allowed us to apply SEM CLEM and TEM-CLEM to further study the direct interactions between TEVs and host cells and its sequelae. WI-38 cells at 2 hours post application (hpa) exhibited both the FM1-43FX and PKH26 fluorescent probes that were used to label TEVs. We analyzed how membrane structures are organized and interact with EV particles from the initial contact through entry to the cell and how the series of events influences the cell structures. We evaluated the quality and validity of data generated from multi-imaging systems used to assess the molecular and structural organization of the cells in qualitative and quantitative manners. The detailed examination of cytoarchitecture in conjunction with fluorescent reporters offered a previously unattainable insight into events occurring on cell surfaces [6].