Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy

Under conventional electron microscopy negatively stained phosphorylase kinase exhibits a bilobal structure resembling two bridged opposing parentheses. In this predominant particle orientation, usually only one bridge is observed; however, in many particles two bridges can be seen. Scanning transmission electron microscopy of unstained phosphorylase kinase shows very similar structures, with a particle mass equivalent to that of the hexadecameric holoenzyme. Partial digestion of the enzyme with chymotrypsin, which preferentially hydrolyzes the alpha-subunits, causes no significant changes in the structure; however, when both the alpha and beta subunits are degraded by trypsin, single lobed particles appear, i.e. the connecting bridges are missing. Mass analysis of scanning transmission electron microscopy images of trypsinized enzyme indicates that the protease does, in fact, split the particle into halves. Transmission electron microscopy of an alpha gamma delta complex isolated after incubation of the holoenzyme with LiBr shows only small particles approximately one-fourth the size of the holoenzyme. Thus, integrity of the beta subunit may be necessary in order for the two lobes of phosphorylase kinase to be bridged. These data also indicate that the subunits are arranged as a bridged dimer of octamers 2 (alpha 2 beta 2 gamma 2 delta 2).     

Sutural mineralization of rat calvaria characterized by atomic-force microscopy and transmission electron microscopy

The application of transmission electron microscopy (TEM) and atomic-force microscopy (AFM) aid the acquisition of detailed structural information on the process of hard tissue formation. The sutural mineralization of rat calvaria is taken as a model for a collagen-related mineralization system. After cryofixation or chemical fixation an anhydrous tissue preparation technique with no staining procedures is used. The atomic-force microscope and the transmission electron microscope are used for structural analysis of the mineralizing region of the sutural tissue. With the application of AFM the collagen macroperiod is shown to be well represented in the unmineralized sutural tissue. At the mineralization front the collagen fibrils are found to be thickened and to change to a characteristic stacked platelet structure. Using TEM the macroperiod is faintly visible before mineral crystallites have formed and is more prominent after the apatite crystallization has started in the fibrils. In this step a needle-like structure of the newly formed apatitic crystals is visible.     

Fixation of platelets for scanning and transmission electron microscopy.

A double fixation method of preparing platelet suspensions for both scanning and transmission electron microscopy is outlined. Prefixation in 0.1% glutaraldehyde allows for immediate preservation of morphologic characteristics induced by experimental procedures, but does not completely destroy platelet surface stickiness. Preservation of surface stickiness allows subsequent production of a platelet pellet for processing for transmission electron microscopy. This pelleting cannot be achieved when higher initial concentrations of glutaraldehyde are used for prefixation. Prefixation in 0.1% glutaraldehyde is also an appropriate initial step for preservation of platelets in suspension for scanning electron microscopy.     

Morphofunctional characteristics of Clara cells in hypoxia in rats from the data of scanning and transmission electron microscopy

The experiments on the model of altitude-chamber hypoxia in rats have established that within 15 days the secretory activity of terminal bronchiolar Clara cells increased in the secret accumulation phase and was accompanied by the transformation of the apical surface relief and ultrastructure of synthetic cell apparatus. Chronic hypoxia lasting for up to 60 days leads to compensation-adaptation changes of Clara cell ultrastructure, providing the intensification of secretion processes and postsecretion repair of membranes of the apical surface cells.     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.     

Shell structure in Difflugia Lobostoma observed by scanning and transmission electron microscopy

Observations by scanning and transmission electron microscopy provide information about shells of Difflugia lobostoma which suggests a complex activity in shell construction. As observed by scanning microscopy, the shell consists of a single layer of sand grains which are organized into rosettes. The sand grains of the rosettes are different in size from those of flat areas between rosettes suggesting that the organism sorts these stones and places them according to size. Hydrofluoric acid treatment dissolves the sand but leaves a web of cement material intact. Examination of such acid treated specimens by transmission microscopy shows structure in the cement material of the shell, and granules of similar structure in the cell body. The rosette pattern observed differs from shell patterns in other species of Difflugia, and this suggests that shell structure may be species specific.     

Ciliary activity in the mouse oviduct as studied by transmission and scanning electron microscopy

The mouse oviduct is covered by dense tracts of ciliated cells interspersed at random with occasional non-ciliated cells. Correlation between scanning electron microscopy and thin section images indicates that in the isolated fimbria most cilia are short (5 µm) and inactive, resting at the end of a uterad-directed effective stroke. These cilia terminate in a 9S−2 tip, the microtubules ending in an electron-dense plaque underneath the cell membrane. At the tip of the cilium a crown of fine extracellular hairs is attached to the ciliary membrane. In the ampulla and isthmus the ciliated cells decrease progressively in number and appear to lie in crypts.     

Use of scanning electron microscopy coupled with transmission electron microscopy in comparative studies of the relation between Schistosoma mansoni and Salmonella typhimurium

Relations between Schistosoma mansoni and Salmonella typhimurium are studied in vivo and in vitro using scanning and transmission electron microscopy as complementary methods. Salmonellae adhesion is a specific process materialized in special places of male and mature schistosome tegumental surface. Interactions are marked by bacterial strong fibres creating a network all around Schistosoma where Salmonellae are dividing. Membrane junction is the last stage leading to symbiotic balance between two biologic systems.     

The crater defect in boar spermatozoa: A correlative study with transmission electron microscopy,scanning electron microscopy,and light microscopy

Nuclear vacuoles resembling the “crater defect” described in bull spermatozoa were observed in 14 boars. Both the incidence of the defect and semen quality were monitored with phase contrast microscopy over a three-month period. The percentages of cratered spermatozoa varied widely both among boars and in ejaculates from the same boar taken on different days. The presence of cratered spermatozoa at a level of 5% or more appeared to be associated with low semen quality. The defect was studied with scanning and transmission electron microscopy and was found to consist of nuclear invaginations, about 0.5 μm in diameter, containing some scanty amorphous electron-dense material. In boars showing a high incidence of spermatozoa with crater defects, abnormalities of the acrosome and perforatorium were common.     

Analysis of fossil bone organic matrix by transmission electron microscopy

A transmission electron microscope (TEM) study was initiated on samples of geological ages ranging from Devonian to Jurassic to analyse the ultrastructure of the organic matrix in fossil bones that have preserved a histological structure after demineralisation. All samples show a network of variably well-preserved fibrils. Within the sampling, the best results were obtained in two specimens: the scales of the Devonian sarcopterygian tetrapodomorph Eustenopteron foordi, and the humerus of Jurassic dinosaur Lappentosaurus madagascariensis. Despite an extended time difference between both specimens, their fossil bone is composed of a plywood-like structure in which the fibrils are very closely packed. These observations support the hypothesis that dense initial packing of collagen fibrils favours the preservation of the fossil bone.     

Observation of symbiote migration in human body lice with scanning and transmission electron microscopy

Bacterial symbiotes in the human body louse Pediculus humanus migrate from the mycetome to the lateral oviducts during the adult molt. Their migration was first described by Ries (E. Ries. 1931. Z. Morphol. Oekol. Tiere, 20:233-367.), who examined sectioned specimens with light microscopy. The present study is a more detailed investigation which involves the use of scanning and transmission electron micrographs. The results of our studies confirm Ries' observations. Micrographs are presented of symbiotes emerging from the mycetome, migrating to the reproductive tract, and invading the lateral oviducts.     

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.