Establishment of embedment-free electron microscopy and analysis of the nature of cytoplasmic matrix by this methodology

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Using polyethylene glycol (PEG), a highly water soluble wax as a transient embedding media, embedment-free sections of chemically fixed tissues are obtained reliably for the electron microscopic examination. After critical-point drying with CO_2, these embedment-free sections present the same aspect of cell ultrastructure as whole mounted, fixed, and dried cultured cells, and filamentous elements (microtrabeculae), which have been unrecognized before in the conventional cytoplasmic matrix, are clearly revealed. Wehen the embedment-free sections are rotaly-replicated with platinum and carbon, resulting images of the sections are of high quality, with excellent resolution and to be quite comparable to those obtained with the rapid freezing, deep etched replica method.With this new methodology, the membrane specializations on the outer and inner cell surfaces as well as the organization of the cytoskeleton is clearly demonstrated. However, after the attainment of the intracellular displacement of cell organelles and some proteins by ultracentrifugation, no marked change in organization of the microtrabecular lattice is discerned.This together with my previous finding that artificial protein solutions at certain concentration exhibit meshworks quite similar to the intracellular microtrabeculae,argue against the idea that gthe filamentous or midrotrabecular strands are the real structure of living cells. Regerdless of the reality or artifact of the microtrabeculae, the PEG method is also applicable to general scanning electron microsoopy, and the intermicroscopic correlation of images between scanning and transmission electron microscopy and light microscopy is easily and reliably performed. Furthermore, the PEG-method is shown to be suitable for light and electron microscopic immunocytochemistry. Therefore this methodology should prove to be valuable adjunct to conventional microscopic techniques.