Figure 3.

Preparations for flat embedding. If cells or tissues are either adherent or can be immobilized onto slides (a), fixation, post-fixation, dehydration, and infiltration with resin are carried out by submerging in cuvettes (b). Thin resin layers are achieved by draining (gravity) (b). For ultra-thin embedding, infiltrated slides are transferred into an acetone-saturated chamber for draining (gravity), which is optionally followed by centrifugation (c). Cells available only as resin infiltrated pellets (e.g. high pressure frozen) can be dropped onto slides and spread/drained in an acetone chamber (d). If resin layers obscure the coordinates of the slides, a stamp can be used for post-embedding labeling for correlative light and electron microscopy (e). Delicate biological samples can be infiltrated within a filter system (f): after light microscopy, samples are placed between acetone resistant filter membranes, sealed in a holder, processed until infiltration by flow through with a syringe. For thin embedding, samples are transferred to a glass slide (for additional LM after polymerization) before draining with filter paper or optional blowing with a dust cleaner for removal of excessive resin (f). For reduction of potential charging effects, specimens are trimmed to a proper size (g), mounted onto aluminum stubs with conductive silver by contacting the glass surface broadly. Specimens are coated with carbon by evaporation (h) and transferred to the focused ion beam (FIB)/scanning electron microscopy (SEM) (i).