Development of a Charge-Resistant Embedding Media for High-Performance Serial Block Face Imaging of Cells and Tissues
Serial block-face scanning electron microscopy (SBEM) enables high-resolution 3D imaging of biological specimens but is often limited by specimen charging due to the use of non-conductive epoxy resins. While heavy metal staining and variable-pressure SEM can reduce charging, these methods compromise resolution or are only partially effective. We report a novel approach using polyethylene glycol (PEG 3350), a water-soluble, non-conductive polymer, as a doping agent to reduce specimen charging without sacrificing imaging quality. Although PEG 3350 alone lacks the mechanical and sectioning properties required for SBEM, we found it can be dissolved in standard Durcupan epoxy resin to improve charge resistance while maintaining physical integrity. Resins doped with 10% PEG 3350 demonstrated a substantial reduction in charging at 1.4-1.8 keV in charge-prone samples such as cultured HeLa cells, lung, and brain tissues, while retaining transparency and sectionability. Unlike other methods, the doped resin performs in high vacuum without compromising signal-to-noise ratio or spatial resolution. Notably, the PEG-doped resin reduced charging at voltages above the typical threshold for undoped resins, which show artifacts above 1.0 keV. Though some charging persisted in lung tissue at 2.0 keV, near-complete mitigation was achieved at slightly lower voltages. Our findings suggest that PEG-doped resins provide a promising route to minimize specimen charging in SBEM, and future studies optimizing PEG molecular weight and concentration could yield a universally charge-resistant embedding medium compatible with high-resolution imaging across diverse sample types.