A new method of preparing embeddment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks.

Abstract

Diethylene glycol distearate is used as a removable embedding medium to produce embeddment-free sections for transmission electron microscopy. The easily cut sections of this material float and form ribbons in a water-filled knife trough and exhibit interference colors that aid in the selection of sections of equal thickness . The images obtained with embeddment-free sections are compared with those from the more conventional epoxyembedded sections, and illustrate that embedding medium can obscure important biological structures, especially protein filament networks. The embeddment-free section methodology is well suited for morphological studies of cytoskeletal preparations obtained by extraction of cells with nonionic detergent in cytoskeletal stabilizing medium. The embeddment-free section also serves to bridge the very different images afforded by embedded sections and unembedded whole mounts .Transmission electron microscopic (TEM)" images of conventional thin sections are influenced by the presence of the embedding material to a degree not often appreciated . The embedding resin, especially epoxy, scatters electrons very much the way the embedded specimen does, rendering it nearly invisible, except where heavy-metal stains are bound (9) . The thin section is most effectively stained at its surface (I1, 18) and is thus essentially a two-dimensional slice of three-dimensional objects whose actual morphology can only be reconstructed with difficulty. Such images formed from exceedingly thin planes are excellent for visualizing many biological structures but are inappropriate for examining three-dimensional networks of protein filaments (5) such as those constituting the cytoskeletal framework (i.e., surface lamina and a protein filament network surrounding the chromatin-containing nucleus : reference 13) .An alternate but under utilized method of electron microscopy, the unembedded whole mount, provides an image 'Abbreviations used in this paper: DGD, diethylene glycol distearate ; MDCK, Madin-Darby canine kidney; nBA, n-butyl alcohol; TEM, transmission electron microscopy . directly from the specimen without resort to heavy-metal stains . In this procedure, the entire fixed, dehydrated, and critical-point dried cell is placed in the electron beam path in vacuo and biological material scatters electrons sufficiently to form very high contrast images. The important differences between stained, embedded sections and unstained whole mounts has been discussed in detail by Wolosewick and Porter (23,24) for unextracted, fixed (i.e., intact) cells .The embeddment-free whole mount has proven essential to the study of the cell architecture remaining after cells have been extracted with nonionic detergent. This procedure removes most lipids and soluble components leaving the cytoskeletal framework (for reviews see references 13 and 14) . This entity, revealed by detergent extraction, retains the configuration of the unextracted cell . Such preparations appear quite empty of structural elements when viewed in ...

Localization of heterogeneous nuclear ribonucleoprotein in the interphase nuclear matrix core filaments and on perichromosomal filaments at mitosis.

“…This is the preparation used for examining mitotic cells. Further extraction with a mixture ofTween 40 Mitotic cells were extracted only with Triton X-100 in CSK buffer, since the compaction of chromatin into chromosomes obviates the need for DNA extraction. To preserve the spindle microtubules, taxol was added to the mitotic cell culture medium 5 min before extraction.…”

Resinless section electron microscopy reveals the yeast cytoskeleton

“…It subsequently was dehydrated, embedded in modified diethylene glycol distearate (supplied by the EMCorp under the name ''Antibed;'' the company no longer exists, and an alternative supplier is being sought) and sectioned to a thickness of 90 nm. The sections were placed on Formvar-covered, carbon-coated copper grids, and the embedding material then removed (9). A JEOL 1200 EXII microscope operated at 80 kV was used to view the resinless sections.…”

“…In contrast, the resinless section can image the entire sample in three dimensions and does not require heavy-metal stains. Also, the support of the sample by the embedding medium has proven unnecessary; once chemically fixed, cell structures are strong and possessed of considerable dimensional stability (8,9).…”

Cytoskeletal arrangement and its intercellular connection in wheat young leaf cells

“…Electron microscopy has long been applied to plant cell research, but why such intercellular connection has not been definitely observed up to now? According to Penman and his colleagues [4], this ignorance may be due to the obscurity caused by embedding medium to biological structures. The definite chemical characteristics of these intercellular cytoskeletons, the way they pass through cell wall, the role they play in plant tissue and the possibility of their developmental change etc, all need further investigation.…”

“…Since then, cytoskeleton became one of the front fields in cell research, and many important findings have been obtained, contributing a great deal to the development of cell biology [3]. However, some important biological structures, especially protein filaments, may be obscured by using the conventional epoxy-embedding medium [4,5]. Furthermore, ultrathin section technique can reveal only 2-dimensional views, so it is difficult to explore the exact organization of whole cytoskeleton.…”