Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: electrostatic charging for section attachment and implementation of an anti-contamination glove box.

摘要: Abstract Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring 3D organization biological samples that are too large to be imaged in an intact state. Producing good quality cryo-sections, however, challenging. Here, we focused on major obstacles success: contamination and around microtome, attachment ribbon sections electron microscopic grid support film. The conventional attaching has involved mechanical force generated by a crude stamping or pressing device, but this disrupts integrity cryo-sections. Furthermore, poor, parts often far from This results specimen instability during image acquisition subsequent difficulty with aligning projection images. have implemented protective glove box surrounding cryo-ultramicrotome reduces humidity within microtome sectioning. We also introduce novel way attach EM film using electrostatic charging. remains place transfer storage devoid related artefacts. illustrate these improvements structure putative cellular 80S ribosomes 50 nm, Saccharomyces cerevisiae .