Two-dimensional Micrographs Research Articles

Fibroblast Foci Are Not Discrete Sites of Lung Injury or Repair

Usual interstitial pneumonia (UIP), the pathologic correlate of idiopathic pulmonary fibrosis, contains characteristic discrete areas of fibroblasts, myofibroblasts, and newly formed collagen, termed "fibroblast foci." These lesions are argued to represent isolated sites of recurrent acute lung injury and suggested to be the mechanism of disease progression. We hypothesized that, rather than isolated, these lesions are part of an organized neoplasm. Morphometric analysis of pentachrome-stained histologic sections of UIP was performed. Using point-counting technique on serial sections, fibroblast foci, arteries, and macrophage clusters were identified and we determined their individual "connectiveness" by estimating the Euler number. Two-dimensional micrographs were collated into a three-dimensional array from which a visual three-dimensional reconstruction could be constructed. Clonality analysis was performed using human androgen receptor gene methylation assay. Blood vessels show significant connectivity with a Euler number of 2, whereas macrophage clusters exhibited no connectivity. The fibroblast foci showed a high level of interconnection with Euler numbers ranging from 19 to 39. The computer generated three-dimensional models provide a visual confirmation of this connectiveness. Human androgen receptor gene methylation assay analysis of the foci showed balanced methylation consistent with polyclonality. The fibroblast foci of UIP are the leading edge of a complex reticulum that is highly interconnected and extends from the pleura into the underlying parenchyma. It is a reactive, rather than a malignant, process.

Using genetic algorithms to evolve three-dimensional microstructures from two-dimensional micrographs

The article describes work to bring together the topics of evolutionary computing and stereology and asks the reader to judge whether such an approach can be genuinely useful or just represents a clever application of computer science. The problem we address is that of constructing three-dimensional (3-D) microstructures from two-dimensional (2-D) micrographs. Our solution is a computer program called MicroConstructor that evolves 3-D discrete computer microstructures, which are statistically equivalent to the 2-D inputs in terms of the microstructural variables of interest. The core of Micro-Constructor is a genetic algorithm that evolves the 3-D microstructure so that its stereological parameters match the 2-D data. MicroConstructor uses a general method of pattern construction, the EmbryoCA, that does not require intervention from the user and is highly evolvable. This article presents initial results from successful experiments to evolve 3-D two-phase microstructures from 2-D input microstructures. The advantages and disadvantages of the method are discussed, and we conclude that the method, though delightfully elegant and full of potential, has yet to prove itself capable of constructing 3-D microstructures that would interest experimentalists and computer modelers.

Evaluation of Electrode Performance Using a Microstructure-Based Simulation Model

The electrochemical performance of electrodes, such as those used in solid oxide fuel cells, depends to a large extent on the microstructure of the electrode material. A modeling approach, based on the lattice Boltzmann simulation method, has been developed to describe the detailed three-dimensional transport and reaction phenomena occurring in a representative sample of porous electrode material. The model geometry is obtained by obtaining statistical information from a two-dimensional micrograph and reconstructing an equivalent three-dimensional structure. The usefulness of this modeling approach is demonstrated by simulating a simplified two-dimensional electrode.

Two-dimensional simulation and modeling in scanning electron microscope imaging and metrology research.

Traditional Monte Carlo modeling of the electron beam-specimen interactions in a scanning electron microscope (SEM) produces information about electron beam penetration and output signal generation at either a single beam-landing location, or multiple landing positions. If the multiple landings lie on a line, the results can be graphed in a line scan-like format. Monte Carlo results formatted as line scans have proven useful in providing one-dimensional information about the sample (e.g., linewidth). When used this way, this process is called forward line scan modeling. In the present work, the concept of image simulation (or the first step in the inverse modeling of images) is introduced where the forward-modeled line scan data are carried one step further to construct theoretical two-dimensional (2-D) micrographs (i.e., theoretical SEM images) for comparison with similar experimentally obtained micrographs. This provides an ability to mimic and closely match theory and experiment using SEM images. Calculated and/or measured libraries of simulated images can be developed with this technique. The library concept will prove to be very useful in the determination of dimensional and other properties of simple structures, such as integrated circuit parts, where the shape of the features is preferably measured from a single top-down image or a line scan. This paper presents one approach to the generation of 2-D simulated images and presents some suggestions as to their application to critical dimension metrology.

An experimental–computational approach to the investigation of damage evolution in discontinuously reinforced aluminum matrix composite

A combined experimental–computational approach to study the evolution of microscopic damage to cause failure in commercial SiC particle reinforced DRAs is dealt with. Determination of aspects of microstructural geometry that are most critical for damage nucleation and evolution forms a motivation for this work. An interrupted testing technique is invoked where the load is halted in the material instability zone, following necking but prior to fracture. Sample microstructures in the severely necked region are microscopically examined in three dimensions using a serial sectioning method. The micrographs are then stacked sequentially on a computer to reconstruct three-dimensional microstructures. Computer simulated equivalent microstructures with elliptical or ellipsoidal particles and cracks are constructed for enhanced efficiency, which are followed by tessellation into meshes of two- and three-dimensional Voronoi cells. Various characterization functions of geometric parameters are generated and sensitivity analysis is conducted to explore the influence of morphological parameters on damage. Micromechanical modeling of two-dimensional micrographs are conducted with the Voronoi cell finite element method (VCFEM). Inferrences on the initiation and propagation of damage are made from the two-dimensional simulations. Finally, the effect of size and characteristic lengths of representative material element (RME) on the extent of damage in the model systems is investigated.

Serial Sectioning Method in the Construction of 3-D Microstructures for Particle-Reinforced MMCs

A serial-sectioning technique is described for obtaining detailed three-dimensional microstructural images from two-dimensional sections of Si-reinforced Al-Si-Mg alloys. Optical micrographs of a series of microstructural sections were generated by the gradual removal of material layers. These micrographs were then enhanced and digitized in a quantitative image analysis system. Three-dimensional microstructure models were then computationally constructed by assembling the digitally acquired two-dimensional micrographs. Serial sectioning was found to be an inexpensive and effective means of accurately depicting heterogeneous microstructures at high resolution. A few relevant observations on material properties also were made during sectioning. Particle cracking was found to occur in larger particles that are located in clustered areas and have their longest dimension aligned with the tensile axis.

Computer modelling of grain microstructure in three dimensions

We present a program that generates the two-dimensional micrographs of a three dimensional grain microstructure. The code utilizes a novel scanning, pixel mapping technique to secure statistical distributions of surface areas, grain sizes, aspect ratios, perimeters, number of nearest neighbors and volumes of the randomly nucleated particles. The program can be used for comparing the existing theories of grain growth, and interpretation of two-dimensional microstructure of three-dimensional samples. Special features have been included to minimize the computation time and resource requirements.

Computer-Assisted Three-Dimensional Reconstruction of Human Dermal Dendrocytes

We attempted to characterize the three-dimensional structure of dermal dendrocytes and to clarify the spatial relationships between dermal dendrocytes and mast cells, macrophages, and nerves. Normal human adult skin (breast, n = 2) was routinely processed for electron microscopy. Every other section (about 50 per data set) was collected at 80-nm intervals traversing about 8 microns of tissue. Grids showing the same cells were photographed by electron microscopy at a magnification of 4000x. Based on the 10-20 photographs per data set, cell outlines were digitized into the reconstruction program at appropriate layers and aligned. Thin, elongated cytoplasmic "dendrites" of dermal dendrocytes in two-dimensional micrographs proved to be thin, membrane-bound flaps in three-dimensional reconstruction. For dermal dendrocytes concentrated about superficial vessels (perivascular dendrocytes), the flaps enshrouded the vessel wall, and for dermal dendrocytes directly beneath the epidermis (subepidermal dendrocytes), these flaps were aligned parallel to the dermal-epidermal junction. The three-dimensional feature of dermal dendrocytes (perivascular and subepidermal) is quite similar to that of perivascular adventitial veil cells, suggesting ultrastructurally identified perivascular dendrocytes and veil cells must be identical cells. In conventional ultrathin sections, 20-40% of perivascular dendrocytes and occasional subepidermal dendrocytes were closely associated with mast cells. When viewed by computer-assisted three-dimensional reconstruction, membrane flaps of dermal dendrocytes consistently shrouded mast cell membranes for 50-90% of their perimeter; mast cells resembled a ball in a baseball glove (dermal dendrocytes). Occasional dermal dendrocytes surrounded non-myelinated nerves in the superficial dermis. Membrane flaps also enabled dermal dendrocytes to present extensive areas to the plasma membranes of adjacent monocyte/macrophages. These findings indicate that dermal dendrocytes are non-dendritic cells that are spatially related to mast cells, monocyte/macrophages, microvessels, and nerves by their membranous flaps. This suggests the need for further study of functional interactions between these cells.

Use of low temperature scanning electron microscopy to observe frozen hydrated seed tissues ofGlycine max

AbstractEvidence is provided to show that frozen, hydrated seeds and seed tissues can be observed by low temperature field emission scanning electron microscopy. This technique allows preparation and observation of seed samples that contain 13–60% water without altering their moisture content. The technique provides information about the surface structure of seeds and also allows specimens to be fractured to reveal internal features of tissues. Futhermore, both halves of fractured specimens can be retained, examined and photographed either as two-dimensional micrographs or as stereo pairs when three-dimensional observation (stereology) or quantitative measurements (stereometry) are desired. Use of this technique avoids artefacts associated with chemical fixation, dehydration, and critical point drying—procedures that are usually required to prepare seed tissues for scanning electron microscope examination. This technique does not affect the degree of hydration in specimens; it can be used to localize water in tissues to determine their degree of hydration. This technique should find wide application in developmental studies of seeds, especially during maturation, imbibition and germination.

Three-dimensional computer modelling system for the study of biological structures.

A three-dimensional computer modelling system has been developed for use in biology, and is currently running on a Sun3 computer. The data originate as a series of two-dimensional micrographs which are digitised via a TV camera. The two-dimensional images are used to select features of interest and to construct a three-dimensional model. This model can be viewed in vector or solid format, it can be rotated about three orthogonal axes and can be viewed in three dimensions as a stereo pair or an anaglyph. The system has been used in a large number of projects over the past 10-15 years, for example, to examine physiological and nerve structures. The time-consuming part of the process is the selection of features, which involves a high level of biological expertise. Present developments are concerned with reduction of the time spent in feature recognition and involve the introduction of expert systems together with human-computer interaction to deal with problems of identification.

Electron microscopic studies of free and proteinase-bound duck ovostatins (ovomacroglobulins). Model of ovostatin structure and its transformation upon proteolysis.

Conformational changes of duck ovostatin (ovomacroglobulin) upon complexing with thermolysin have been studied by electron microscopy. Both free and thermolysin-bound ovostatin preparations were negatively stained with uranyl acetate, a series of three pictures were taken at 10 degrees specimen tilt intervals (+10 degrees, 0 degrees, and -10 degrees), and images of the inhibitor molecules were observed in three dimensions. Four approximately cylindrical subunits were observed in free ovostatin. Two subunits associated approximately midway from both ends to form a dimer of four arms. Two dimers associated with each other at the midpoint to form a tetramer. The proteinase susceptible "bait" regions were located near the center of the molecule. Eight arms of the tetramer take various configurations. The orthogonal extent of free tetrameric ovostatin in a two-dimensional micrograph averages 26.0 +/- 4.7 x 34.0 +/- 5.0 nm. Upon complexing with thermolysin, all eight arms curl toward the center of the molecule, having four arms upward and the other four downward. Thus, proteinase-bound ovostatin has a uniform structure with a 2-fold axis of symmetry. The overall structure of the complex is more compact with average dimensions of 16.9 +/- 0.6 x 16.9 +/- 0.6 x 19.9 +/- 0.4 nm. From these electron microscopic studies we propose that a proteinase reaches to the center of the free ovostatin molecule and attacks the bait region. Subsequent to proteolysis the subunit arms curl and entrap the enzyme within the ovostatin molecule. The results support the unique mechanism of inhibition of proteinases by alpha 2-macroglobulin and ovostatin postulated from biochemical observations (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724; Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498).

Three-dimensional imagery and quantitative analysis in SEM studies of nematodes

During the past 20 years, the application of scanning electron microscopy (SEM) to nematological investigations has considerably increased our knowledge about the structural features of nematodes. However, two relatively new techniques, namely three-dimensional visualization of specimens (stereopsis) and taking quantitative measurements from three-dimensional images (stereometry) offer additional insight. This investigation demonstrates how these techniques can be utilized and applied to studies in nematology. Stereopsis of nematodes is easily attained with a standard SEM by tilting the specimen 4 to 8° between two successive micrographs. The resulting micrographs, which constitute a stereo pair, contain parallax information that is needed for stereopsis. Three-dimensional visualization of the pair is achieved by an individual using a simple stereo viewer or by an audience when a Polaroid projection system is employed. The three-dimensional images that result improve resolution and clarify the shapes, topography and spatial relationships of structural features. Quantitative information, which is not attainable from a single two-dimensional micrograph, can be obtained from a stereo image by using a four-minor stereoscope equipped with a parallax measuring system. Information obtainable with this instrument allows one to make simple, accurate measurements, contour maps and models of specimens. The application of these two techniques, which are used in conjunction with SEM, are potentially important to morphological, anatomical, and taxonomic studies of nematodes as well as to their interactions with host tissues and naturally occurring pathogens.

Image enhancement of aggregate aerosols by stereopsis

Irregularly shaped aggregate aerosols from several sources were studied by stereopsis. The metallic oxide and soot aerosols studied here had structures ranging from simple stacked arrangements to complex aggregates and branched structures. Images from two electron micrographs of the same area were taken at 0° and 10° from the horizontal axis and used for stereographic visualization. Stereopsis aids in reliable determination of the number of primary particles present in an aggregate, and a more realistic appreciation of the shape of complex aggregates. It was found that the number of primary particles counted in large samples of diesel particles was 36 ± 5% greater using stereopsis as opposed to conventional viewing of two-dimensional micrographs.

A Relatively Inexpensive Microprocessor-Linked Digital Plamimeter for Electron Microscopic Morphometry

Stereology provides a theoretical basis for powerful morphometric methods for the estimation of three-dimensional structural parameters from two-dimensional electron micrographs of cells and tissues. These methods assume at the start that one has a sufficiently large set of micrographs containing valid structural data. The task of obtaining from these micrographs the large quantity of data needed to get statistically valid results has been eased in two general ways. Sampling of data in the micrograph can be done rapidly by point and intersection counting methods. An alternate method, planimetry, obtains all the data in the micrograph, but in general is more time-consuming than point and intersection counting. Some of the relative inefficiency of planimetry is compensated when a digital planimeter is coupled with a computer. Areas and lengths can be computed simultaneously as fast as profiles are traced. Furthermore, rapid and numerically accurate compilation and statistical analysis of the data can be done automatically as the planimetry is done, not as a separate step after the data have been obtained.

Influence of Electron Noise on Three-dimensional Image Reconstruction

Abstract A three-dimensional reconstruction requires the same integral dose as a conventional two-dimensional micrograph provided that the level of significance and the resolution are identical. The necessary dose D for one of the K projections in a reconstruction series is, therefore, the integral dose divided by K.

Image Reconstruction Applied to High Voltage Microscopy

Greatly increased specimen penetration, which is the principle advantage of high voltage electron microscopy, carries with it an increased need for techniques to interpret the large amount of three-dimensional information projected into two-dimensional micrographs. Stereo views can provide very useful information and are widely used. However, for the general specimen, stereo views are limited in their ability to produce quantitative results. At the high voltage microscope facility, Univ. of Wisconsin, we have begun a program to develop and apply three dimensional reconstruction techniques to the microscopy of thick specimens.