Comparison between direct methods for determination of microbial cell volume: electron microscopy and electronic particle sizing.

Size frequency distributions of different phototrophic and heterotrophic microorganisms were determined by means of scanning and transmission electron microscopy and electronic particle sizing. Statistically significant differences existed among the three techniques used in this study. Cells processed for electron microscopy showed lower mean cellular volumes than those processed for electronic particle sizing, reflecting a shrinkage by factors ranging from 1.1 to 6.2 (mean, 2.3). Processing of cells for scanning electron microscopy caused higher shrinkage than processing for transmission electron microscopy. Shrinkage was dependent neither on the size nor on the cell wall type of the microorganism. When processed for scanning electron microscopy, phototrophic bacteria were strongly shrunken, whereas heterotrophic microorganisms were less affected. A direct relationship existed among phototrophic bacteria between percentage of shrinkage and specific pigment content. This was probably a consequence of the pigment extraction by organic solvents during the dehydration process, previous to the critical point drying, necessary to examine the specimens under the scanning electron microscope.     

Improved transmission electron microscopy technique for the study of cytologic material

A modification of the technique of Coleman et al for the preparation of single cells in cytologic specimens for electron microscopy (EM) is described. By employing materials in the initial cytologic processing that are useful for EM, such as a paraformaldehyde-glutaraldehyde fixative, lactated Ringer's solution as a rinsing medium, glycerol as a mounting medium and cacodylate buffer for removal of coverslips, the use of alcohol fixatives and standard mounting media could be avoided. This preserved the cytoplasmic detail, which is usually degenerated in cells removed from cytologic specimens and processed for EM.     

Combined immunofluorescence and field emission scanning electron microscope study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl-methyl-methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross- bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.     

Electron microscopy of cells showing viral cytopathic effects in Papanicolaou smears

A technique is described whereby cells showing cytologic changes suggestive of virus infection by light microscopy can be processed further for examination in the electron microscope so that virus particles present in the cell can be visualized directly. We present the results of electron microscopy of over 100 Papanicolaou-stained smears processed this way. The morphologic changes in the cells and the ultrastructural appearances of the virus particles are demonstrated. This technique is particularly valuable for retrospective studies of mounted cytologic or histologic material. It has also proven to be a valuable research tool in the study of human polyomavirus and human wart virus infection.     

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.     

Application of the membrane filter technique to bromodeoxyuridine immunochemistry for exfoliative cytology

The membrane filter technique for smear specimens of tumors in bromodeoxyuridine (BrdU) immunochemistry is described. The staining results of Raji cells processed using the filter technique was compared with that obtained by the conventional cytospin method. Although the BrdU mean labeling index (LI) for in cytospin specimens was almost the same as the LI in membrane filter specimens, filter specimens showed excellent staining and less cell destruction compared with those processed by cytospin. Small amounts of tumor specimens such as squamous cell carcinoma and polymorphous low-grade adenocarcinoma also were processed using the membrane filter appliance. For squamous cell carcinoma, the LI for the filter specimens was 5.36 ± 0.38 and that of the paraffin sections was 5.56 ± 0.38. The membrane filter technique provided relatively undamaged specimens for exfoliative cytology and will be useful for immunohistochemical evaluation of tumor cells and for routine, noninvasive cytological screening.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

Cytochemical demonstration of increased phospholipid content in cell membranes in chlorphentermine-induced phospholipidosis

We recently introduced a novel cytochemical approach to high-resolution cytochemistry of phospholipids in biological tissues. The technique consists of adsorption of bee venom phospholipase A2 to colloidal gold particles (PLA2-gold complex) and subsequent application of this complex for localization of the enzyme substrate, i.e., glycerophospholipids. In the present study, this technique was applied at the post-embedding level, in both light (LM) and transmission electron microscopy (TEM), to investigate drug-induced phospholipidosis, an experimental disorder in which the lysosomal catabolism of phospholipids is inhibited. Rats received one week of daily treatment (40 mg IP/kg) with chlorphentermine (CP), a cationic amphiphilic drug known to induce phospholipidosis in several tissues. Glutaraldehyde- and osmium-fixed lung and kidney tissues from both treated and control animals, were embedded in Epon and sections processed for labeling by PLA2-gold. In CP-treated specimens the presence of large osmiophilic inclusions in several cell types of lung parenchyma and kidney cortex confirmed the onset of phospholipidosis. These inclusions were densely labeled by PLA2-gold at both LM and TEM levels. Two general types of abnormal inclusions were distinguished on the basis of their ultrastructure and labeling pattern by PLA2-gold, suggesting different content or configuration of phospholipids. Moreover, quantitative evaluation of labeling density over various membrane compartments in lung alveolar cells evidenced significantly increased phospholipid content after CP treatment. In type II pneumocytes, such increases were measured in membranes of the RER, Golgi complex, outer and inner nuclear envelope, and the basolateral and apical domains of the plasma membrane. In capillary endothelial cells, the basal and luminal domains of the plasma membrane also showed an increase in labeling density. These results further demonstrate the potential usefulness of the PLA2-gold technique for in situ ultrastructural localization of phospholipids in normal and pathological tissues.     

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.     

A preliminary evaluation of moist environment ambient temperature scanning electron microscopy

A technique which allows scanning electron microscopy of untreated fresh biological specimens in a moist environment at ambient temperature, is described. It requires virtually no preparation of the specimens prior to insertion in the microscope. This technique, referred to as Moist Environment Ambient Temperature Scanning Electron Microscopy (MEAT SEM) is evaluated in relation to the conventional scanning electron microscopy and optical microscopy. Results obtained on conidia of Aspergillus niger, tomato fruit cortical cells (Lycopersicon esculentum) and epidermal cells of perianth segments of Narcissus sp., suggest that MEAT SEM is a promising technique for obtaining good quality micrographs of biological tissues in conditions nearest to those in vivo.     

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.     

Subcellular distribution of the alpha subunit(s) of Gi: visualization by immunofluorescent and immunogold labeling.

The subcellular distribution of the alpha subunit(s) of Gi has an obvious bearing on the ability of this protein to interact with receptors and targets and on its potential to serve in still unexplored capacities. In this study, we have examined the distribution of Gi alpha by means of light and electron microscopy. The cells employed were mouse 3T3 fibroblasts, normal rat kidney fibroblasts, rat C6 glioma cells, human umbilical vein endothelial cells, and human 293 kidney fibroblasts. By indirect immunofluorescence, two patterns of Gi alpha were evident. The more prominent was that associated with phase-dense, cytoplasmic structures exhibiting a tubule-like morphology. A similar distribution was noted for mitochondria, indicating attachment to a subset of microtubules. The second pattern appeared as a diffuse, particulate fluorescence associated with the plasma membrane. By immunogold labeling and electron microscopy, two populations of Gi alpha were again evident. In this instance, labeling of the plasma membrane was the more prominent. Gold particles were most often evenly distributed along the plasma membrane and were concentrated along microspikes. The second, less abundant population of Gi alpha represented the subunit (or fragments) within lysosomes. Specificity in immunolabeling was confirmed in all instances by immunotransfer blotting, the use of antibodies differing in specificities for epitopes within Gi alpha, the absence of labeling with preimmune sera, and the decrease in labeling after preincubation of antisera with appropriate peptides. These results support the proposal that several populations of Gi alpha exist: those evident within the cytoplasm by immunofluorescence, those present at the plasma membrane, and those evident within lysosomes by immunogold labeling.     

Localization of avidin in virus-transformed and damaged chicken embryo fibroblasts by immunofluorescence and the avidin-biotin-peroxidase complex (ABC) technique

Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction.     

Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.     

New approaches to the study of sphingolipid enriched membrane domains: the use of electron microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture. To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3 x 10(-7) M [1-(3)H]sphingosine. [1-(3)H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-(3)H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy. Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.     

Orthogonal arrays of intramembrane particles in the supporting cells of the guinea-pig vestibular sensory epithelium

Membrane specializations of the contact region between afferent nerve endings and supporting cells of the sensory epithelia of guinea-pig vestibular endorgans were examined by thin-section and freeze-fracture electron microscopy. The calyx-type nerve endings (C-endings) are separated from supporting cells (SC) by a 25-30 nm space. At irregular intervals along the upper lateral surface of supporting cells, the intercellular space narrows markedly to form special close contacts between the C-ending and SC plasma membranes. Freeze-fracture replicas reveal membrane specializations--orthogonal arrays of particulate units--in the region where the close intercellular contacts were found in sections. Orthogonal arrays consisting of from 5 to 20 units were observed on the cytoplasmic (P) fracture face of the lateral SC plasma membrane. These particulate units from a 12 x 12-nm square, and each unit is composed of four 6-nm subunits. Possible roles of the orthogonal arrays are discussed.     

Scanning electron microscopy of plasma etched implant specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Endocrine cells of the human gastric mucosa

Summary By light and electron microscopy investigation of the human gastric mucosa five types of ultrastructurally different endocrine cells have been detected: 5-hydroxytryptamine storing enterochromaffin (EC) cells, gastrin storing G cells, and functionally undefined ECL, D and D₁ cells. By direct application of Masson's argentaffin reaction as well as of Sevier-Munger's and Grimelius' argyrophil method to electron microscopy specimens, selective deposition of silver grains upon the endocrine granules of such cells was obtained. In particular, only EC cell granules reacted to the argentaffin method, granules of both EC and ECL cells heavily reacted to Sevier-Munger's technique, granules of EC, ECL, G and D₁ cells reacted to Grimelius' technique, while D cell granules failed to react either to argentaffin or argyrophil methods. By the application of the same silver methods to paraffin sections as well as by other selective staining methods for endocrine granules (5-hydroxytryptamine techniques, lead-haematoxylin, HCl-basic dye method), at least four of the above cell types were also identified under light microscope. This opens the way for extensive studies of such cells in conventional histologie specimens.This investigation was supported in part by grant N.70.01022.04 from the Italian Consiglio Nazionale delle Ricerche.