Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy

The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by scanning tunneling microscopy (STM). STM is a near field technique that can resolve structures at the nanometer level and thus can image individual molecules. Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and globular aggregates. The linear arrays consist of side by side dimers with the long axis of the dimer perpendicular to the aggregated chain. Individual molecules of phosphorylase kinase appear to be planar, bilobate structures with a 2-fold axis of symmetry and a central depression.     

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.     

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.     

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.     

Analysis of Mason-Pfizer monkey virus Gag particles by scanning transmission electron microscopy

Mason-Pfizer monkey virus immature capsids selected from the cytoplasm of baculovirus-infected cells were imaged by scanning transmission electron microscopy. The masses of individual selected Gag particles were measured, and the average mass corresponded to 1,900 to 2,100 Gag polyproteins per particle. A large variation in Gag particle mass was observed within each population measured.     

Heterogeneity of Escherichia coli ribosomes established by scanning transmission electron microscopy.

Quantitative mass image analysis of Escherichia coli ribosomal particles by scanning transmission electron microscopy (STEM) provided direct evidence that presumably homogeneous preparations of ribosomes are, in reality, populations of heterogeneous particles. Variations in composition, relative molecular mass (Mr) and shape were observed both in the monosomes and in the ribosomal subunits. None of these changes can be resolved visually; they can be evaluated only by computer processing. The variations in relative mass and shape monitored by values of radius of gyration (RG) were attributed to the loss of ribosomal proteins and/or factors and correlated with the changes in ribosome composition and biological activity. The highest activity was found in monosomes prepared from the standard 0.5 M NH4Cl wash. With increasing concentrations (up to 1.5 M) of NH4Cl in the wash buffer the activity decreased slowly, then dropped rapidly to about half in 2 M NH4Cl. The most striking effects were observed in ribosomal particles washed with 0.1 M NH4Cl. The 70S monosomes and the 30S subunits attained maximum Mr and RG values (2660 kDa and 76 A, and 990 kDa and 75 A, respectively), which were greater than the theoretical values, while the activity was minimal (approximately 12%). The Mr and RG parameters of the 50S subunits remained uneffected by the NH4Cl washes (approximately 1600 kDa and 68 A).     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.     

The rat pituitary cleft: A correlated study by scanning and transmission electron microscopy

Summary SEM reveals that the inner surface of the pituitary cleft is lined by a continuous layer of marginal cells possessing microvillous and ciliated apical surfaces. The ciliated cells are more numerous on the posterior side (toward the pars intermedia) than on the anterior side of the cleft (toward the pars distalis). In contrast small infoldings (crypts) were occasionally noted only on the marginal layer covering the distal part of the hypophysis. In some areas of the cleft the surface features of the marginal cells are rather similar to the epithelial cells populating the upper parts of the respiratory tract in their topography and distribution. In other regions they also show striking similarities with the ependymal cells (tanycytes) lining the lateral recesses of the 3rd ventricle and the infundibular process with which the pituitary cleft has a very close topographical relationship.The parenchymal cells of the pars distalis are closely related to the flattened marginal cells of the cleft. The intercellular spaces of the pars distalis form a three-dimensional labyrinthic series of cavities continuous with the submarginal spaces of the cleft. Further SEM and TEM results demonstrate that the majority of the microvillous marginal cells lining both sides of the cleft possess surface features such as bulbous protrusions, laminar evaginations and large cytoplasmatic vacuoles, which are very likely the expression of an active transport of fluids.On the basis of these results it is concluded that the fluid-like material (colloid) present in the pituitary cleft is mainly derived from the fluids contained in the lacunar spaces of the pars distalis. Thus, marginal cells by absorbing fluids from the cleft by active endocytosis, may transport to the pars intermedia material (or hormones) produced in the distal part of the gland and vice versa.The cilia present on many marginal cells, based on their 9+2 tubular pattern, possess a kynetic role. This is very similar to that shown by the ciliated cells of the ependyma lining the brain ventricles. The occurrence of ciliated cells within the pituitary parenchyma (mainly in the follicles) suggests that they probably arise from the ciliated cells populating the marginal layer of the cleft and with which the parenchyma cells are closely related.     

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.     

Membrane contents of distinct subpopulations of coated vesicles determined by scanning transmission electron microscopy

In order to investigate the heterogeneity of clathrin-coated vesicles purified from rat liver, and to quantitate rigorously their membrane contents, we have analyzed scanning transmission electron micrographs of unstained coated vesicles before and after extraction with the non-ionic detergent Triton X-100, as well as of vesicles whose coats had been removed by dialysis against 10 mM or 100 mM Tris (pH 8.2). Their respective distributions of particle masses were thus determined and compared, in light of complementary biochemical quantitations of lipid and protein. Smaller coated particles, 25-45 MDa in mass and 60-80 nm in diameter, lose no mass when extracted with Triton, and disappear when their coats are dissociated. We conclude that they do not contain membrane vesicles, although they have dense, presumably proteinaceous, cores. They may represent particles generated during tissue homogenization or, possibly, a storage form of clathrin. The remaining 70% contain bona fide vesicles: these particles are 75-150 nm in diameter, and their average mass is about 80 MDa, of which 48 MDa is contributed by coat proteins, 10-12 MDa by phospholipid and cholesterol, and 20-22 MDa by vesicle-associated proteins. Their vesicles are of two types: smaller, denser, vesicles that contain substantial amounts of internalized material, and larger, less dense, vesicles that do not. The distinction between them may, in view of other findings, reflect a difference between coated vesicles derived respectively from the Golgi and the plasma membrane.