Transmission electron microscopy of tissue prepared for scanning electron microscopy by ethanol-cryofracturing.

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of specimens cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.     

Morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy

Isotretinoin ingestion during the first trimester of human pregnancy can induce malformations of the skull, ears, face, central nervous system, eyes, palate, lungs, circulatory system, limbs, and digits. A single oral dose of isotretinoin on day 8 of gestation in hamsters induces a similar syndrome of congenital malformation. The present study concerned scanning electron microscopic (SEM) observation of embryonic and fetal hamster craniofacial structures at 4, 8, 12, 24, 48, and 72 hr after administration of an oral dose of 50 mg/kg isotretinoin or an equivalent volume of the vehicle. The variability in development among control embryos recovered 4 hr after treatment precluded objective assessment of pathologic change by SEM at very early time points. Craniofacial damage was obvious within 8-12 hr of isotretinoin treatment, and it included hypoplasia of the maxillary and mandibular processes of the first branchial arch, a rudimentary second arch, and apparent collapse of the forebrain. Equivalent fusion between the lateral nasal process and the maxillary process and between the medial nasal process and the maxillary process in treated and control embryos accounts for the very low incidence of cleft lip observed in fetuses. The terminal microstomia was not associated with excessive merging or overgrowth of the first arch components. Hypoplasia of the first arch can account for retinoid-induced macrostomia and microstomia.     

A simplified method for preparing rotifer trophi for scanning electron microscopy

A simple, quick technique for the preparation of rotifer trophi for scanning electron microscopy is described. The method permits visual monitoring of the extraction process and does not require critical point drying of the specimens. Micrographs showing fine, structural detail of the hard parts of the mastax of representatives of the following genera are presented:Asplanchna, Conochilus, Filinia, Hexarthra, Keratella, Proalides, Synchaeta, andTrichocerca.     

A method for preparing and staining histological sections containing titanium implants for light microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

A new method of preparing fish chromosomes for scanning electron microscopy

A preparative ion-etching technique has been developed which enhances the images of fish chromosomes obtained by scanning electron microscopy.     

Submicroscopic organization of M. pneumoniae studied by means of negative contrasting, ultrathin sections and scanning electron microscopy]

The ultrastructure of M. pneumoniae, grown on a solid culture medium and in a liquid one, was studied by a number of methods. Two types of cells were shown to prevail in the culture: spherical cells (0.5--1 micrometer) forming chains of different configurations and filamentous cells (5 micrometer long and greater) with spherical enlargements along their whole length. The absence of microcapsules made M. pneumoniae different from other species of mycoplasms, and the organism proliferated by division into 2 daughter cells, equal or unequal in size, by the segmentation of the cytoplasm and the formation of elementary bodies inside the cell and on its surface.     

A new method of preparing insects for scanning electron microscopy.

Acidified 2,2-dimethoxypropane (DMP), used as an alternative to regular fixation and dehydration methods for insects, was found to be the only successful means of preparing the sugarbeet root maggot larva, Tetanops myopaeformis (R?der) (Diptera:Otitidae), for the scanning electron microscope. No morphological changes were evident when DMP treated sugarbeet root maggot adults were compared to fresh (unfixed) adults and glutaraldehyde-osmium tetroxide fixed adults. The method has been used with success on several gropus of insects. Acidified CMP is quickly hydrolyzed by water in tissue to acetone and methanol. DMP is advantageous in that it penetrates water impermeable cuticles rapidly and saves several steps and time in the fixation and dehydration process.     

A new method for isolating pollen and spores from acetate peels for scanning electron microscopy

A procedure for precisely extracting pollen and spores from cellulose acetate peels made from Carboniferous permineralizations is described. This technique produces either whole or sectioned clean grains and allows for the correlation of morphological and ultrastructural features by scanning electron microscopy. The critical examination of pollen and spores from peels prepared for earlier studies is now possible using this technique.     

A rapid method for resectioning of semithin large epoxy sections for electron microscopy

A simple and rapid method is described for resectioning semithin large epoxy tissue sections. Six-micron thick sections are cut, floated on a polystyrene coverslip, and stained with toluidine blue. The coverslip is trimmed to the size of a BEEM capsule cap and placed section face-up inside the cap. A BEEM capsule with the conical end cut off is fitted onto the cap, filled with plastic and polymerized. The capsule and coverslip are trimmed off and thin sectioning is performed. This method allows the study of multiple tissue lesions in their entirety, i.e. serially from beginning to end, without sacrificing any part during trimming as in conventional thin sectioning.     

The morphology of cell lines suitable for vaccine production studied by scanning electron microscopy]

Five nontumorogenic cell lines suitable for vaccine production were studied by SEM. It was shown that diploid cell line 41 originated from sheep embryo kidney and also two heteroploid cell lines, line 4921 originated from embryo skin and muscles of the African green young monkey and line 4647 from kidney of the adult monkey, maintained normal cell morphology and normal growth pattern in early and in later passages in cultures. Some alterations in epithelial dense monolayer formation were revealed in the heteroploid cell lines: in line 455 originated from spleen of the adult African green monkey, and in line 4184 originated from line 41. The revealed alterations can be considered as the early morphological signs of the transformation of epithelial cells in culture. These cell lines also retained the stability of their morphological characteristics at the earlier and later passages. All the studied cell lines were free of contaminating agents.     

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.