Scanning microscopy. Supplement最新文献

Low-angle diamond knives, one of them with an angle as low as 14.6 degrees, have been tested to measure compression induced during the sectioning of polystyrene latex spheres embedded in epoxy resins. Compression diminishes roughly proportional to the sectioning angle (sum of knife and clearance angles). The beneficial application of low-angle diamond knives in biology and material science is illustrated by sections through a muscle fiber (compression reduced by a factor of 3), sections through a collagen fiber (meridional resolution improved by a factor of 2) and sections through a copper grid bar (reduced curling). These low-angle diamond knives are as resistant as the classical knives having angles of 45 degrees and are routinely used by the author. Low-angle diamond knives with angles down to 35 degrees are now commercially available.

Nucleic acid sequences can be localized on chromosomes in the electron microscope after hybridization with a biotinylated DNA probe followed by detection with a primary antibiotin antibody and a secondary antibody coupled to colloidal gold. Hybridization probes can also be labelled with alternative ligands such as N-acetoxy-2-acetylaminofluorene (AAF), Dinitrophenyl-dUTP and Digoxigenin-dUTP. Multiple labelling is possible if these differently modified DNA probes are used in conjunction with colloidal gold preparations of varying particle sizes. A substantial signal amplification can be achieved by incubating preparations with successive cycles of primary antibiotin antibody followed by a biotinylated secondary antibody. Detection is with Streptavidin-gold, and in the case of highly and moderately repeated sequences, the signal is visible in the light microscope. Detailed protocols are given for EM in-situ hybridization to whole mount metaphase chromosomes and include instructions necessary to perform multiple sequence localization and signal amplification.

The preparation of stable protein-gold conjugates is important in the quantitative colloidal gold staining. The protein adsorption onto colloidal gold particles and pH-dependent stability of protein-gold conjugates were examined using albumin and fibrinogen as model proteins. Albumin stabilized gold sols at pH values higher than 5.0. The amount of albumin necessary to stabilize gold sols was lowest at pH 5.6 and slightly increased as the pH of the adsorption medium was increased. Albumin was also able to stabilize gold particles even at acidic pH values ranging from 3.5 to 4.5, if a sufficient amount of albumin was added. Stable fibrinogen-gold conjugates were obtained at pH values above 6.5. Unlike albumin, the same amount of fibrinogen was able to stabilize gold sols as pH was increased from 7 to 10. The stability of the prepared protein-gold conjugates was very sensitive to the pH of the storing medium. The protein-gold conjugates were least stable at the isoelectric pH of the protein. The results suggest that protein-gold conjugates should be prepared at pH where conjugates are most stable, instead of pH close to the isoelectric point.

A surface preparation method for the SEM based on cryofixation is presented suitable for the demonstration of membrane particles on whole cells. LLC-PK1 cells (a renal epithelial cell line in culture) were fast frozen, freeze substituted, critical point dried, shadowed with 2 nm of platinum carbon and stabilized with a carbon backing layer. Membrane bound particles are visualized by the material dependent backscattered electron image mainly originating from the platinum shadow. The surface of the LLC-PK1 cells is almost free of precipitated material indicating that the culture medium is removed during freeze substitution or critical point drying. The apical plasma membrane with microvilli and ciliae is well preserved and differences in particle density can be detected. The feasibility of the coating technique for backscattered electron imaging was tested on the well known hexagonally arranged intramembranous particles of fractured and partially freeze dried yeast. This 16.5 nm periodic structure is clearly demonstrable on the bulk SEM-specimen stablized with the carbon backing layer. Without a carbon layer severe shrinking artifacts occurred.

Plastic transverse sections of labeled semithick and ultrathin cryosections prepared by the Tokuyasu procedure demonstrate that section surfaces can be smooth or rough, sometimes showing fracture-like structures. These surface irregularities can arise from specimen properties, sectioning/fracturing and compression during sectioning, loss of cytoplasmic material during labeling, and from the final drying step. Nevertheless, small and sensitive marker molecules like 4 nm gold-protein A, ferritin-IgG and 1 nm gold-IgG bind predominantly to the cryosection surface of even mildly fixed specimens as long as the section is well preserved. In our experiments, marker penetration is enhanced by section damage. Marker incubation of ultrathin cryosections obtained by cryo transverse sectioning of IgG-labeled semithick cryosections shows also that penetration of uncoupled antibodies into cytoplasm and nuclei of chicken erythrocytes is limited.

To study the possible effects of chemical fixation upon antigenicity and structural preservation, the subcellular localization of LamB-LacZ hybrid proteins in Escherichia coli K-12 strains pop3234 and pop3299 was investigated both by cryo-ultramicrotomy and freeze-substitution. Immuno-gold labelling of sections of freeze-substituted bacteria showed the same localization of the hybrid protein as found after cryo-ultramicrotomy. The efficiency of labelling of the accumulated form of the hybrid protein was lower after freeze-substitution whereas the efficiency of labelling of the membrane-bound form showed no difference. Different fixatives and Lowicryl resins had no clear effect on the label-efficiency but the complex substitution medium, containing osmium tetroxide, uranyl acetate and glutaraldehyde, in combination with the apolar Lowicryl HM20 gave the best sectioning properties and membrane contrast. For this specific problem, although the somewhat better preservation after freeze-substitution, cryo-ultramicrotomy is to be favored since it is much less time-consuming, there are no freezing problems, ultrastructural preservation is sufficient and the theoretical benefits of freeze-substitution are not expressed.

3T3 and HeLa cells, grown as a monolayer, have been rapidly frozen by propane jet as a fresh preparation, without pretreatment. In some experiments the frozen cells were fractured at -170 degrees C, thawed into fixative and viewed by high-resolution SEM after critical-point drying. In other experiments the frozen cells were thawed into fixative unfractured. These preparations were refrozen in 15% methanol, fractured and deep-etched for replication and TEM study. The technique used in this work appears to give rapid rewarming from -170 degrees C to 0 degree C with little evidence of ice crystal growth. The cells fractured before thawing, examined by SEM, show extensive extraction of both nucleus and cytoplasm with deep views of nuclear chromatin, and of cytoplasmic organelles caught amongst rather distorted filaments of the cytoskeleton. Initial fixation for the SEM work was light (0.3% glutaraldehyde for 10 mins) so that structure is seen as it would be retained for antibody labelling.

The heterogeneity in human neutrophil granules was examined by the ultrastructural localization of a series of antigens which have been previously identified with neutrophil granules by either physical separation or biochemical/biological techniques. All samples were prepared by cryofixation and molecular distillation drying (LifeCell Process), a two-step physical method that achieves cryofixation by metal mirror freezing and drying by the controlled, incremental heating of cryofixed samples in an ultrahigh vacuum. After drying, the samples were either exposed to vapor-phase osmium followed by embedment in Spurr resin, or they were exposed to formaldehyde vapor followed by embedment in Araldite resin. An indirect streptavidincolloidal gold procedure was used for immunoelectron microscopy on ultrathin sections. Subcellular ultrastructural morphology of neutrophils prepared by this method was good compared to standard electron microscopic techniques and superior compared to comparable, published electron microscopic cryomethods applied to neutrophils. Immunogold localization of myeloperoxidase, cathepsin G, lysozyme, lactoferrin, beta 2-microglobulin, and CD-15 antigens showed high intensity and specificity of labeling in the intracellular granules. Patterns of labeling varied from antigen to antigen, demonstrating granule heterogeneity both within and among neutrophils. This methodology is useful in the exploration and definition of granule heterogeneity and function.

It is desirable to examine the cytobiology of cell adhesion to the same materials which are contemplated for use in biomedical and biotechnological devices. It is also of fundamental interest to examine adhesion to substrates with properties which are likely to influence adhesion in controlled ways. In many of these applications the materials of choice are polyurethane elastomers due to their physical properties and resistance to biodegradation. Polyurethanes have a two phase microstructure consisting of hydrophilic hard segments and hydrophobic soft segment domains. Variations of both the chemistry and the morphology of these microdomains may be produced. It is well understood that the hydrophilic/hydrophobic nature of surfaces affects cellular adhesion and the adsorption of extracellular proteins. Since polyurethane microdomains have dimensions in the range of 10-100 nm, hence the size of proteins and cell-surface receptors, polyurethane microdomain structure could influence order at the cell-material interface. Polyurethanes may be prepared as thin films with excellent properties for use as specimen supports in High Voltage transmission Electron Microscopy (HVEM) at 1 MeV. This permits the imaging of the cytoskeleton and other internal features of whole mounts of adherent cells, rather than tedious thin sectioning required for conventional TEM. Subsequently the surface morphology of these preparations may be imaged with high resolution SEM. Finally, the polyurethane itself may be stained and imaged by either HVEM or high resolution SEM in order to relate polyurethane micro-morphology to cellular features.