A new method for comparative light and electron microscopic studies of individual cells, selected in the living state

Abstract

SUMMARY A method is described which permits comparative light and electronmicroscopic studies of cell cultures, cell spreads or single selected cells which have been kept in the Plastic Film Dish (PFD). The PFD is a versatile large surface tissue culture chamber which, for electron microscopy, is mounted with a transparent FEP‐Teflon film bottom. Cells are observed, selected and marked on the PFD‐bottom with a high power inverted light microscope. The cells are fixed and dehydrated with a semi‐automatic device while they are still in situ in the PFD. During the preparation steps for electron microscopy the topographical relationship between individual cells and between cells and cell support is accurately retained. After embedding and polymerization the Teflon film is easily peeled off the polymerized Epon, leaving a replica of the mark around the selected cell. This permits relocation of the selected cells for ultrathin sectioning in a plane plan‐parallel to the original cell support. To enable orientated sectioning of selected cells in a plane perpendicular to the cell support, cells are tagged with Letraset‐letters after original embedding and polymerization. Subsequently the re‐embedded polymerized specimens are orientated in the microtome in a position which permits controlled thin sectioning of the tagged cells in the previously selected plane.

A simple and efficient method for preparation of isolated ovarian follicles for transmission electron microscopy

“…Note the granulosa cell nucleus (GCN) and the abundant mitochondria (M) populating the cytoplasm. Note the junction between the 2 granulosa cells (astrix) membranes such as Teflon [21,22] and polycarbonate [23] have been used for preparation of individual cells for TEM, but not for isolated ovarian follicles. Polycarbonate membranes require staining for cell visualization.…”

“…Alternatively, since the surface of all biological cells carry a negative charge, the cells can be adhered to poly-Lysine coated glass cover slips [16] or charged nylon membranes [17]. Individual cells can be also prepared for TEM in situ on supporting membranes like plastic [18,19], cellulose esters [20], Teflon [21,22] and polycarbonate [23].…”

Concerning the nature of the cryosectioning process

“…For the embedding of freeze dried FTS the cutting procedure was slightly modified. The FTS were collected on a 5 pm Epon section and positioned in the middle of a transfer letter ' o' , which was attached to the 5 pm section to facilitate retrieval after embedding (see also Van Ewijk & Hosli, 1975). The FTS were pressed, freeze dried and fixed in osmium tetroxide vapour as described earlier.…”

A resin slide technique to select fixed embedded cells for transmission electron microscopy

“…The selection of particular cells or groups of cells within a block of embedded material is an important aspect of many cytological investigations. In some cases it is possible to observe cells directly at the edge of the block, and trim accordingly (Codling & Mitchell, 1976;Ellis, 1971;Nelson & Flaxman, 1973;van Ewijk & Hosli, 1975). To observe the cells more clearly under the light microscope, and to locate cells within the interior of the block, it is necessary to cut thick sections.…”