Correlative light, scanning and Transmission Electron Microscopy of polyethylene glycol (PEG) embedded and subsequently de-embedded tissues

Abstract

Our understanding of the detailed morphology of bioloqical specimens often requires the correlation between images of the surface by scanning electron microscopy (SEM) and of the underlining interior structure by light microscopy (LM) or transmission electron microscopy (TEM). In order to accomplish the correlation with high quality, the polyethylene glycol (PEG) embedding and subsequent deembedding has been revealed to be a simple and quite reliable procedure.Tissue or cell samples are fixed and dehydrated in ethanol according to the conventional procedures. Vials containing the specimens in 100% ethanol are warmed in an oven at about 60°C and approximately equal amount of pure molten PEG-4000 is added in the vials. PEG is easily dissolved by gentle agitation. Thereafter the specimens are transferred to pure molten PEG contained in well-dried gelatin capsules. After the specimens have been sunk to the bottom of the capsule, each capsule is immersed in liquid nitrogen to solidify PEG. Portions of the solidified PEG including the specimens are cut out with a razer blade and mounted with dental wax on supporting stubs fitting the collet of the microtome. The specimen blocks are sectioned with a well-dried glass or diamond knife. Wrinkle-free sections, 200-300 nm thick, can be easily obtained with some practice unless the room atmosphere is humid. The sections are picked up with a platinum loop filled with 2.5 % sucrose and mounted on glass slides for LM or formvar-coated grids for TEM, both of which have previously been treated with 0.1 % poly-L-lysine. For LM sections may be processed for immunohistochemistry. For TEM, sections on grids are put into a submerged grid holder in 90 % ethanol. The holder is transferred to 100 % ethanol and subsequently to a critical point apparatus with carbon dioxide. For SEM, the specimen blocks with its mirror-smooth face, after taking sections, is removed from the stubs and immersed in warm water to get rid of PEG. The blocks are then dehydrated in ethanol and critical point dried with carbon dioxide.