PubMed Health⌕ Search

Biomedical subjects

A Leith

Publications and source records attributed to A Leith.

16 recordsLinked to original sources
PubMed2001

High-resolution CryoFESEM of individual cell adhesion molecules (CAMs) in the glycocalyx of human platelets: detection of P-selectin (CD62P), GPI-IX complex (CD42A/CD42B alpha,B beta), and integrin GPIIbIIIa (CD41/CD61) by immunogold labeling and stereo imaging.

The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b alpha,b beta), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809-819, 2001)

Antigens, CD↗
PubMed2000

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome.

BACKGROUND: This study addresses the general problem of dividing a density map of a nucleic-acid-protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches approximately 3 A it is generally possible to interpret its shape (i. e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. RESULTS: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 A resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. CONCLUSIONS: The new method, when applied to an 11.5 A cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.

Bacterial Proteins↗
PubMed1999

Cubic membrane structure in amoeba (Chaos carolinensis) mitochondria determined by electron microscopic tomography.

Cubic membranes occur in a variety of membrane-bound organelles in many cell types. By transmission electron microscopy (TEM) these membrane systems appear to consist of highly curved periodic surfaces that fit mathematical models analogous to those used to describe lipidic cubic phases. For the first time, a naturally occurring cubic membrane system has been reconstructed in three dimensions by electron microscopic tomography, and its periodicity directly characterized. Double-tilt tomographic reconstruction of mitochondria in the amoeba, Chaos carolinensis, confirms that their cristae (inner membrane infoldings) have the cubic structure suggested by modeling studies based on thin-section TEM images. Analysis of the membrane surfaces in the reconstruction reveals the connectivity of the internal compartments within the mitochondria. In the cubic regions, the matrix is highly condensed and confined to a continuous, small space between adjacent cristal membranes. The cristae form large, undulating cisternae that communicate with the peripheral (inner membrane) compartment through narrow tubular segments as seen in other types of mitochondria. The cubic periodicity of these mitochondrial membranes provides an ideal specimen for measuring geometrical distortions in biological electron tomography. It may also prove to be a useful model system for studies of the correlation of cristae-matrix organization with mitochondrial activity.

Amoeba↗
PubMed1997

Tinkerbell--a tool for interactive segmentation of 3D data.

A software system for interactive manipulation of three-dimensional data has been developed, based on the Open Inventor tool kit. The primary use of this software system is in the segmentation of tomographic reconstructions of subcellular structures. To this end, the reconstruction is represented by volume rendering and displayed in stereo. A three-dimensional cursor with adjustable shape and size is used to define and isolate regions of interest inside the volume, based on the user's expert knowledge. Once isolated, the region of interest can be conveniently analyzed and displayed.

Animals↗
PubMed1996

Direct visualization of A-, P-, and E-site transfer RNAs in the Escherichia coli ribosome.

Transfer RNA (tRNA) molecules play a crucial role in protein biosynthesis in all organisms. Their interactions with ribosomes mediate the translation of genetic messages into polypeptides. Three tRNAs bound to the Escherichia coli 70S ribosome were visualized directly with cryoelectron microscopy and three-dimensional reconstruction. The detailed arrangement of A- and P-site tRNAs inferred from this study allows localization of the sites for anticodon interaction and peptide bond formation on the ribosome.

Anticodon↗
PubMed1991

Confocal microscopy and three-dimensional reconstruction of electrophysiologically identified neurons in thick brain slices.

Three-dimensional morphology and electrophysiology were correlated from individual neurons in a thick brain slice preparation. The hippocampal formation from immature and adult rats was cut transverse to the longitudinal axis into 500 microns-thick slices which were maintained under physiologic conditions. Individual neurons were impaled and physiologically characterized using microelectrodes. Recordings were made from the soma and in some cases from a dendrite. The impaled neurons were filled through the microelectrode with the fluorescent dye lucifer yellow and imaged by confocal scanning laser microscopy using an analog preprocessor. As many as 180 optical sections were recorded as a function of depth through the slices. Images are presented as a series of optical sections, stereo pairs, or three-dimensional reconstructions. Both stereo contouring and volume rendering methods were employed, and the reconstructions were viewed from any arbitrary perspective. Dendritic and axonal fields were separated from each other and displayed separately or as different pseudocolors. The three-dimensional reconstructions provided perspectives that were difficult or impossible to appreciate by viewing the optical sections or conventionally formed stereo pairs.

Animals↗
PubMed1991

The use of confocal microscopy and STERECON reconstructions in the analysis of sea urchin embryonic cell division.

A laser scanning confocal microscope has been used to investigate the development of the sea urchin embryo. The samples were fixed in Carnoy's solution at various developmental stages, stained for DNA with the Feulgen reaction, and optically sectioned with a BioRad MRC-500 confocal microscope. Computer-generated stereographic projection images and a three-dimensional contour tracing and reconstruction system were employed to investigate the cleavage pattern during the 6th cleavage division. Cell division is found to be asynchronous during the 6th cleavage, with macromere derivatives completing division first, followed by mesomeres, and finally by the outer quartet of micromeres (which begins division only after macromeres and mesomeres have completed their respective divisions). Sixth cleavage produces an embryo comprising 60 cells. Asynchronous division was also observed within individual tiers of blastomeres. Variations in the orientations of cell division axes within individual tiers of cells were also observed. The utility of computer-graphics reconstruction techniques for both quantitative and qualitative developmental analysis are discussed.

Animals↗
PubMed1991

Organelle rearrangement and cell volume changes during squeezing invasion of peritoneal elastic lamina by targeted murine breast carcinoma cells.

Murine breast cancer cell lines were developed to selectively invade the peritoneum while they proliferated in ascites form in the abdominal cavity. In a dominant form of invasion, tumor cells showed special affinity for elastin fibers and squeezed through narrow gaps in the elastic fiber meshwork of the stroma. Even in fixed tissue, such cells could be recognized as being in the process of invasive migration because of their dumbbell shape. This appearance was similar to that of diapedetic blood cells traversing bone marrow sinus endothelium. Three-dimensional STERECON graphics reconstruction from serial thick sections of 44 such cells was carried out. The reconstructions showed that, in mid-penetration, the cells spread extensively over the exterior surface of the elastic fiber meshwork. The cell surface contact of these forward projections was mainly with the elastic fiber outer coat of microfibrils, but small areas of the cell surface also fused directly to inner-core elastin. The morphological rearrangement of the cytoskeleton was minimal in both types of attachment areas. The location of these forward facing attachments is consistent with mechanisms for pulling the invasive cell through the gap. Lamellopodia formation and clustering of cytoplasmic organelles occurred more commonly at the forward-facing part of the cell. Morphometry of the reconstructions showed that a contraction of the whole cell occurred during the squeezing/migration process suggestive of an additional pushing process. However, our invasive cell lines showed marked differences in the degree of cell shrinkage. The process of adhesion and squeezing of tumor cells through elastin meshworks in vivo is clearly a complex phenomenon. Changes in cell surface activity appear to play a significant role in establishing the necessary 'foothold' component of invasion and, possibly, in the generation of tractive force as well.

Animals↗
PubMed1990

The relative merits of direct morphometry of reconstructions of whole cells, and statistical morphometry by stereology of random sections of cells.

Stereology, or the derivation of quantitative, three-dimensional (3-D) data about cells by statistical analysis of the structures of random sections, is widely used in cytology and pathology. However, there are situations where this approach is inadequate, and only an analysis of a homogeneous population of whole cells will give the required results. This involved 3-D reconstruction from physical or optical sections, or tomography or photogrammetry of whole-cell mounts. Use of stereo views of individual sections or projections adds considerably to the information available for both contouring and reconstruction. Recent image-processing advances in clinical radiography have shown, for the first time, that rapid, high-resolution digitization and contrast enhancement enable nearly all structural details to be routinely extracted from the micrographs and adequately portrayed. Three-D whole-cell reconstructions provide the digital data for many kinds of morphometric measurements on both whole cells and their individual organelles and membranes. Rapid fixation or freezing allows improved quantitative structure/function correlations of organelles with disturbances in cell metabolism or gene expression.

Animals↗
PubMed1988

Three-dimensional reconstruction of cells from serial sections and whole-cell mounts using multilevel contouring of stereo micrographs.

A comprehensive computer-graphics-based system (STERECON) is described for tracing and digitizing contours from individual or stereopair electron micrographs. The contours are drawn in parallel planes within the micrographs. Provision is also made for tracing and digitizing in full three-dimensional (3-D) coordinates in any direction along linear structures such as cytoskeletal elements. The stereopair micrographs are viewed in combination with the contours being traced on a graphics terminal monitor. This is done either by projecting original electron micrograph (EM) negatives onto a screen and optically combining these images with contour lines being drawn on the monitor, or by first digitizing the images and displaying them directly on the monitor along with the contour lines. Prior image digitization allows computer enhancement of the structures to be contoured. Correction and alignment routines are included to deal with variable section thickness, section distortion and mass loss, variations in photography in the electron microscope, and terminal screen curvature when combining projected images with contour lines on the monitor. The STERECON system organizes and displays the digitized data from successive sections as a 3-D reconstruction. Reconstructions can be viewed in any orientation as contour stacks with hidden lines removed; as wire-frame models; or as shaded, solid models with variable lighting, transparency, and reflectivity. Volumes and surface areas of the reconstructed objects can be determined. Particular attention was paid to making the system convenient for the biological user. Users are given a choice of three different stereo-viewing methods.

Animals↗
PubMed1979

Role of aggregation factor and cell type in sponge cell adhesion.

A pair of sponge species, Microciona prolifera and Halichondria bowerbanki, which lack mutual species specificity in their aggregation "factor", are useful in establishing the mechanisms of action of these factors. These sponges were dissociated both mechanically, which leaves the factor on the cell surface, and by Humphrey's (1963) method, which isolates the factor from the cells. The adhesive specificities which arose, in the various combinations tested, point to an intercellular factor bridge consisting of a single symmetrical unit. An analysis of most other workers' results is consistent with this interpretation. However, MacLennan and Dodd's (1967) results using other species would require a bridge consisting of two or more asymmetrical units. Differences were found in the specificity of adhesion of various types of cells within a single species. This presents a heretofore unconsidered problem in assesing the adhesive factor's mechanism of action. Three structurally distinct cell types were separated from a suspension of dissociated Microciona cells by velocity sedimentation. These cells differ greatly in adhesiveness. The differences in adhesion are correlated with numbers and positions of cells incorporated into aggregates. Such differences are considered in explaining the mechanism of action of the factors.

Animals↗
PubMedDate not supplied

Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction.

To define the ultrastructural accommodation of mineral crystals by collagen fibrils and other organic matrix components during vertebrate calcification, electron microscopic 3-D reconstructions were generated from the normally mineralizing leg tendons from the domestic turkey, Meleagris gallopavo. Embedded specimens containing initial collagen mineralizing sites were cut into 0.5-micron-thick sections and viewed and photographed at 1.0 MV in the Albany AEI-EM7 high-voltage electron microscope. Tomographic 3-D reconstructions were computed from a 2 degree tilt series of micrographs taken over a minimum angular range of +/- 60 degrees. Reconstructions of longitudinal tendon profiles confirm the presence of irregularly shaped mineral platelets, whose crystallographic c-axes are oriented generally parallel to one another and directed along the collagen long axes. The reconstructions also corroborate observations of a variable crystal length (up to 170 nm measured along crystallographic c-axes), the presence of crystals initially in either the hole or overlap zones of collagen, and crystal growth in the c-axis direction beyond these zones into adjacent overlap and other hole regions. Tomography shows for the first time that crystal width varies (30-45 nm) but crystal thickness is uniform (approximately 4-6 nm at the resolution limit of tomography); more crystals are located in the collagen hole zones than in the overlap regions at the earliest stages of tendon mineralization; the crystallographic c-axes of the platelets lie within +/- 15-20 degrees of one another rather than being perfectly parallel; adjacent platelets are spatially separated by a minimum of 4.2 +/- 1.0 nm; crystals apparently fuse in coplanar alignment to form larger platelets; development of crystals in width occurs to dimensions beyond single collagen hole zones; and a thin envelope of organic origin may be present along or just beneath the surfaces of individual mineral platelets. Implicit in the results is that the formation of crystals occurs at different sites and times by independent nucleation events in local regions of collagen. These data provide the first direct visual evidence from 3-D imaging describing the size, shape, orientation, and growth of mineral crystals in association with collagen of a normally mineralizing vertebrate tissue. They support concepts that c-axial crystal growth is unhindered by collage hole zone dimensions, that crystals are organized in the tendon in a series of generally parallel platelets, and that crystal growth in width across collagen fibrils may follow channels or grooves formed by adjacent hole zones in register.

Animals↗
PubMedDate not supplied

Sterecon--three-dimensional reconstructions from stereoscopic contouring.

Sterecon is a system for making 3-D reconstructions or measurements by tracing from stereopair images. The stereopair images may come directly from a microscope, such as a transmission or scanning electron microscope. Alternatively, the images may be created from a stack of thin slices, such as a confocal light microscopy depth series, an electron tomographic volume, or a set of serial histological slices. When the structure to be studied is thick or complex, a serial stack of stereoscopic images can be used. Objects are traced within the images, and their coordinates are entered into a line or contour database. The contour database can be used for 3-D structure measurement, and the contours can be displayed as a reconstruction. Sterecon has interfaces from other software which can generate the input images and to other software for further display and analysis.

Animals↗
PubMedDate not supplied

SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields.

The SPIDER system has evolved into a comprehensive tool set for image processing, making use of modern graphics interfacing in the VMS and UNIX environment. SPIDER and WEB handle the complementary tasks of batch processing and visualization of the results. The emphasis of the SPIDER system remains in the area of single particle averaging and reconstruction, although a variety of other application areas have been added. Novel features are a suite of operations relating to the determination, modeling, and correction of the contrast transfer function and the availability of the entire documentation in hypertext format.

Animals↗