Staining for Serial Blockface SEM Imaging

Sample preparation is that main part of the Large Volume 3DEM workflow that may frighten people who have not done biological EM before. It is largely a matter of following a recipe, and using appropriate protection.

The same staining method works for serial blockface SEM, FIB-SEM, and will work for traditional single slice TEM and massive-scale multiTEM imaging system. Most people use roughly the same method – that presented by Tom Deerinck and others presented as a poster at Microscopy and Microanalysis in 2010 (described here, with their protocol here).   Our own variation on this approach will also be posted here.

Remember EM Cores can stain tissue for you.

A couple of comments though are worth noting before starting.

What is staining so important? Heavy metal staining is a critical technique in EM because normal tissues contain little that can be seen by EM.   Tissues are thus stained with osmium, which binds double bonds in membrane fatty acids, uranyl ions to bind lipids and carbohydrates, and lead which binds lipid and protein, and greatly enhances prior osmium staining. Detailed explanations can be found here (Hua ref).

In traditional TEM, sections are usually cut and then stained.  Metal ions penetrate into the plastic section and stain membranes and protein.  These heavy atoms deflect incident electrons and prevent them from passing through the section, creating a shadow in the image.  Traditional biological SEM relies on a coating of gold or other material being deposited (sputter coated)  on sample surface to create secondary electrons, which are detected and create an image of the sample surface.

In contrast,  in SBF-SEM, heavy metal stains must be introduced into the sample prior to embedding in plastic resin (en bloc staining), and the depth to which they penetrate limits the sample depth to which imaging that can be performed.   As the block -face is exposed during imaging, the electron beam interacts with these heavy nuclei, curving their trajectories and causing them to be ejected from the sample. Ejected – or backscattered electrons – then hit the BSE detector and so provide the signal that generates the image.

In SBF-SEM, metal staining is doubly important as it also provide a route for electrons out of the imaging area, dispersing them within the sample.  Charging artifacts – like hazy imaging, drifting, etc – are much worse with poorly or sparsely stained tissue.  In practice, if you have poor staining, the images look muddy, are noisy (salt and pepper), require a lot more scanning time to image, and may exhibit image drift, cloudy backgrounds, and accentuate knife chatter marks, scratches, and debris deposition may all be accentuated.