A protocol for Epon-embedding-based correlative super-resolution light and electron microscopy

This protocol describes a detailed step-by-step sample preparation procedure for Epon based post-embedding correlative super-resolution light and electron microscopy (SR-CLEM). The newly developed fluorescent protein, mEosEM, is the core in this protocol. The advantage of this method is to simultaneously obtain high-quality LM and EM images from the same ultrathin section of Epon-embedded sample after conventional chemical fixation.

Ultracentrifugation deforms unfixed influenza A virions

Negatively stained influenza virions sometimes show irregular morphology and are often referred to as pleomorphic. However, this irregular morphology has not been visualized when ultrathin-section transmission and scanning electron microscopies are used. This study focused on the effects of ultracentrifugation on influenza A virion morphology, as negative staining often involves ultracentrifugation to concentrate or purify virions. The morphologies of unfixed, glutaraldehyde-fixed and osmium tetroxide-fixed virions were quantitatively compared before and after ultracentrifugation, and it was found that, without chemical fixation, approximately 30 % of virions were altered from oval to irregular shapes following ultracentrifugation. By contrast, most glutaraldehyde-fixed virions remained uniformly elliptical, even after ultracentrifugation. When a virus with an 11 aa deletion at the C terminus of its M2 cytoplasmic tail was ultracentrifuged, its morphology was appreciably deformed compared with that of the wild-type virus. These results demonstrate that the native morphology of influenza A virions is regular but is disrupted by ultracentrifugation, and that the cytoplasmic tail of M2 is important for virion integrity.