Eleven tungsten atom cluster labels: high-resolution, site-specific probes for electron microscopy.

Two derivatives of a tungstate cluster containing 11 tungsten atoms (W11PO39SiR4-) have been synthesized which enable them to be covalently attached to biomolecules at specific sites. The tungstate cluster is 1.0 nm in diameter, electron dense, and visible in the electron microscope. One derivative is a W11-sulfonyl chloride, reactive with amines and sulfhydryls. The second compound is a W11-thiosulfonate which can be used to label sulfhydryl groups. These new labels are beam resistant and provide significantly higher resolution then most other electron microscopy (EM) markers. Labeling of the protein albumin is described as an example.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

Actin filament labels for localizing protein components in large complexes viewed by electron microscopy

Localizing specific components in three-dimensional reconstructions of protein complexes visualized in an electron microscope increases the scientific value of those structures. Subunits are often identified within the complex by labeling; however, unless the label produces directly visible features, it must be detected by computational comparison with unlabeled complex. To bypass this step, we generated a cloneable tag from the actin-nucleating protein Spire that produces a directly visible “pointer” to the subunit after actin polymerization. We have used this new label to identify the intron of the C complex spliceosome to its small domain by fusing the 10 kDa Spire moiety to the affinity label that binds recombinant stem loops in the pre-mRNA substrate and assembling an actin filament on the particle.     

Inorganic phosphors as new luminescent labels for immunocytochemistry and time-resolved microscopy

A new strongly luminescent marker consisting of inorganic crystals is described for time-resolved microscopy. These crystals, known as phosphors, show delayed luminescence, unlike prompt fluorescent labels such as FITC, TRITC and phycobiliproteins, and are therefore potentially suitable for time-resolved microscopy. The luminescence of these phosphors is strong and non-fading in comparison to FITC/TRITC, and not significantly influenced by pH or temperature. The phosphor yttriumoxisulfide activated with europium emits maximally at 620 nm with a typical half life-time of approximately 700 musec, upon excitation with near ultraviolet light (360 nm). Phosphors for immunocytochemical staining were made by ball milling and were stabilized in suspension with polycarboxylic acids. Proteins such as avidin, protein A or immunoglobulins were allowed to adsorb to the surface of the phosphors. The immunocytochemical properties of the conjugates were evaluated in a model system of latex beads with defined surface antigens and in a cellular system containing fixed human lymphocytes or erythrocytes. Specific cytochemical staining was observed in suspension as well as on glass slides. A specially constructed time-resolved microscope was used to suppress the fast decaying fluorescence, thereby permitting visualization of the specific, slowly decaying luminescence of the phosphor label without the necessity of integration. Finally, the use of multiple phosphors with different kinetic and spectral characteristics for multiparameter studies is indicated.     

Polyethyleneimine as tracer particle for (immuno) electron microscopy.

Polyethyleneimine (PEI) is proposed as a tracer for use in electron microscopical investigations. Relative small molecules are available (molecular weight 600-60,000). PEI is soluble in water; it is not visible in the electron microscope without further treatment, but can easily be detected as a particle by contrastting it with phosphotungstic acid or OsO4. Using PEI of a molecular weight of 40,000, particles of 10 nm diameter can be produced. The strong cationic character of PEI results in electrostatical binding to anionic sites. Hence perfusion and immersion of tissues with PEI of various molecular weights offers possibilities to either study the location of anionic sites or pathways of transport. Anionic sites could be demonstrated in the normal and pathologic glomerular basement membrane. Work on the use of PEI as a marker particle in immunoelectronmicroscopy is in progress.     

Lanthanum as an intracellular stain for electron microscopy

Summary Results obtained after the normal aldehyde fixation of duodenal enterocytes for electron microscopy have been compared with results obtained when 0.1% Malachite Green or 10mm lanthanum chloride had been added during aldehyde fixation. Sections were examined without further staining, and after counterstaining with lead citrate and uranyl acetate. In unstained sections, lanthanum-treated material showed improved contrast when compared to results from the other two methods. Also, after counterstaining, areas showing excellent contrast were much more frequent and more readily detected in the lanthanum-treated material. In the microvilli of enterocytes fixed in the presence of lanthanum, the plasmalemma-glycocalyx was defined more clearly and the results were more pleasing subjectively. When Malachite Green was present in the fixative, good contrast was observed more frequently than in routinely fixed tissues, but less often than in those treated with lanthanum. It is suggested that the addition of lanthanum chloride or Malachite Green to the fixative may prove useful in many ultrastructural studies.     

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.     

Monoclonal antibodies against calcitonin. Characterization and application in light and electron microscopy immunocytochemistry

Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.     

Inorganic trimetaphosphatase as a histochemical marker for lysosomes in light and electron microscopy.

A new cytochemical method is presented for the light and electron microscopic localization of lysosomes in mineralized and soft tissues. Inorganic trimetaphosphate is used as substrate in a lead chelate incubation medium at pH 3.9. Lysosomes in several tissues are strongly reactive, and reaction product is frequently present in Golgi saccules and GERL. The reaction can be differentiated from acid glycerophosphatase activity, is relatively insensitive to fixation and demineralization procedures, and the reaction is often complete after short incubation times.     

Water soluble polyacrylamide as embedding medium for electron microscopy

We have developed a method for embedding biological materials for electron microscopy in the water soluble polymer polyacrylamide (PAM). The fixed samples are infiltrated with an aqueous solution of acrylamide, which is then polymerized to form a hydrated gel. The gel is ready for sectioning after insolubilization by cross-linking with glutaraldehyde. Water is retained throughout the whole embedding process and contact of the samples with organic solvents is completely avoided. Sections are best stained with silicotungstic acid. The potential of this technique has been evaluated by detailed examination of several representative tissues. Observation of the native periodicity of 17 nm in PAM embedded myelin indicates a very good structural preservation. The method gives optimal results with samples which are easily infiltrated, such as pellets of cells.     

Structure of trichomonads as revealed by scanning electron microscopy.

A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné, Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal; emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The early spermatid nuclei of the grasshopper, Acrida lata, have been observed electron microscopically. The irregularly compact chromatin mass appears closely attached to the nuclear envelope. This mass migrates subsequently into a more central portion. It seems to participate in the formation of the nucleolus as a nucleolar organizer. At the time when the chromatin mass and frequently the nucleolus undergo involution, clusters of peculiar granular bodies 130 m in average diameter and 200 A wide filamentous elements among the bodies make their appearance in the nucleoplasm. The particles constituting the granular bodies are composed of DNA, but their matrix consists of RNA. The term microkaryosome is proposed for such granular body, because it is similar in chemical components to karyosome, but the former is smaller in size than the latter. It is suggested that the microkaryosome may be related with the paracrystalline formation of nucleoprotein.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The first indication of differentiation of the Jensen's ring has been detected in an early stage of spermiogenesis of Felis catus Linné when the pair of centrioles takes up a position immediately beneath the plasma membrane. The chromatoid bodies appear in the early spermatid cytoplasm through the nuclear pore complex. In a more advanced stage, such bodies have been found in association with the striated columns, the distal centriole or the proximal part of flagellum and the Jensen's ring. As the spermiogenesis proceeds, the bodies have decreased their size and density, and finally disappear in mature spermatozoa. The chromatoid bodies seem, therefore, to share with the centriole the capacity to form the connecting piece. As a consequence of disorganization of triplet microtubules of the centriole, a noticeable material appears in the center of lumen of the centriole to be identifiable as a distinct precursor of the central pair of axonemal complex. Microtubules are first developed as the sheath of principal piece of the sperm flagellum, originating from the plasma membrane surrounding the axonemal complex.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The fine structure of the spermatids in late stages of the differentiation, which appeared in the testis of early pupa of the silkworm Bombyx mori Linné, was studied in the electron imcroscope, being fixed in buffered (pH 8.2) 2.5% osmium tetroxide or 3% potassium permanganate.The clear band differentiates into elaborated elements consisting of an array of at least 12 membranes which run loosely winding along the major axis of the spermatid. The elaborated clear band, i. e., clear band derivatives, may be an apparatus to facilitate the activity of spermatozoa, since they are present along the full length of the remarkably elongated premature spermatozoa.It has been revealed that the clear band derivatives possess a highly ordered, fine structure which is seen to be of a paracrystalline nature. The periodic pattern has first occurred along the long axis of the clear band derivatives. After such structure is decomposed into an apparently homogeneous material, a characteristic periodic pattern occurs again crossed the major axis of the clear band derivatives, the significance of such ultrastructural changes remaining obscure.The tubular structure appears through the head part of the developing spermatids, revealing even in an apical region where the nucleus is not visible, and it appears enlarged at the base of the nucleus, but no more visible in the tail piece. In the stages when the clear band becomes progressively specialized, the tubular structure appears attached to the nucleus, although it situated at the peripheral part of the cell in a more early stage of the differentiation. The tubule is incompletely separated into two layers by a dense septum projected from the tubular wall, suggesting that such structure provides the tubule with a relatively large interior surface for metabolic reactions.This study was supported by Grant GM-8327-04 from the United States Public Health Service.