Three-dimensional graphical reconstruction from HVEM stereoviews of biological specimens by means of a microcomputer

A microcomputer reconstruction technique has been developed in order to permit a larger exploitation of stereomicroscopy. The microcomputer facility consists of a digitizing tablet, a microcomputer, a graphics terminal, a graphics plotter and a printer. The technique has been applied to the study of HVEM stereopairs, performed by recording two images of the same area of a specimen (thick section of araldite-embedded leech ganglion neurons), tilted relative to the beam axis through an angle 0/20 degrees. Coordinates of N conjugate points of interest, expressed in a common reference system were obtained with the help of a digitizing tablet and the misorientation between the two images was determined by a method based on a least square technique. New projections of the object on different planes are provided by the microcomputer facility. Also the microcomputer method permits to obtain new stereopairs drawings, in various orientations and slices from a three-dimensional reconstruction of the object oriented in any direction in space. The method permits to obtain computed anaglyph drawings, printed here, which are stereoviews of the same object.     

A method for determining the crystal orientation for any tilt position when using a transmission electron microscope double-tilt specimen holder

A method is described in this short communiction which provides a rapid and precise determination of the crystal specimen orientation in any tilt position when using a doule-tilt holder in the transmission electron microscope (TEM). It is necessary to establish three pairs of tilt angles (α_i, β_i) (i = 1, 2, 3) where three recognized directions of the crystal specimen are aligned to the electron beam direction respectively, in order to determine the crystal specimen orientation for any tilt position in a double-tilt specimen holder. In addition, the tilt position (α, β), where any orientation [ u v w] is aligned to the beam direction, can be determined with the aid of this method.A software system for computer applications of the above method has beed developed.     

Block-Surface Staining

A method is described by which the tissue exposed on sectioning a specimen embedded in paraffin can be visualized in situ. The fixed specimen is impregnated with lead acetate, dehydrated in dioxane, infiltrated with paraffin and embedded. Tissues exposed on sectioning are developed by applying to the cut surface of the block a solution of potassium sulphide in water. Concentrations of the reagents used and the time intervals for the procedure are dependent upon the size of the specimen and upon the degree of contrast required. The method is described as it was applied to the study of a small human fetus in cross section. Representative photographs are included to show the results obtained.     

Determining effective centroid position in biomechanical testing: a technique for simplifying whole bone analysis

BACKGROUND: Whole bone in vitro biomechanical compressive testing can be complicated by three factors: sample asymmetry, heterogeneous material properties, and unknown effective centroid location. METHOD OF APPROACH: The technique presented here facilitates the calculation of effective centroid position, modulus of elasticity and equivalent uniform strain magnitude for a cross section of bone from a simple whole bone compressive test. Simplification of section response to load is achieved through a combination of linear beam and simple planer geometry theory. The technique requires three longitudinal strain gauges be affixed around the test specimen cross section of interest, gauge position need not be determined. Sample loading is then accomplished using a simple four point loading jig. RESULTS: Results of the technique are presented on an object with known elasticity and geometry, an aluminium tube, and seven pairs of equine third metacarpal whole bones. CONCLUSIONS: Average cross section modulus of elasticity, equivalent uniform cross section strain, and effective centroid locations were all predicted to within the range of published values. Employing the testing setup and analysis technique presented in this paper resulted in a significant savings in both implementation complexity and cost over previously available techniques.     

High energy X-ray scattering tomography applied to bone

High energy synchrotron X-ray scattering was developed for reconstruction of specimen cross-sections. The technique was applied to a model specimen of cortical bone containing a capillary tube of silicon, and reconstructions were produced with either full diffraction rings or texture-related subsets of a given ring. The carbonated apatite (cAp) 00.2 and 22.2 reconstructions and the Si 311 reconstructions agreed with absorption-based reconstructions from the measured X-ray transmissivity recorded during diffraction pattern acquisition and from reconstructions produced subsequently of the same specimen using a commercial microCT (microComputed Tomography) scanner.     

Tree fern growth strategy in the Late Devonian cladoxylopsid species Pietzschia levis from the study of its stem and root system

Portions of stems from five new anatomically preserved specimens of Pietzschia levis from a new Late Devonian plant locality of eastern Tafilalt, Anti-Atlas (Morocco), were analyzed to complete the preliminary reconstruction previously done with a single specimen. The basal part of the longest new specimen consists of an obconical portion of stem surrounded by a thick mantle of adventitious roots. Roots are connected to the peripheral strands of primary xylem specific to the stele of Pietzschia stems. Roots grow outwardly; they cross the cortex and the broad central pith at a steep angle and emerge from the stem lower down. The number of roots produced at one level increases conspicuously from the base towards the distal end of the obconical portion of stem. By contrast, cross-sectional dimensions of roots at their origin level decrease distally. Individual roots increase in diameter, and their stele gets more lobed as they grow through stem tissues. The large number of roots at the specimen base and their wider dimensions at this level contribute to the conspicuous enlargement of the stem base. Patterns assessed from the reconstruction of the Pietzschia levis root system may be close to those of the older cladoxylopsids Pseudosporochnales comprising an upright trunk. Growth strategies in the small-statured species P. levis and in younger arborescent ferns of the Psaronius type are compared. They differ mainly in the relative lengths of epidogenetic vs. apoxogenetic growth phases of the stem.     

Microfocal X-ray CT imaging and pulmonary arterial distensibility in excised rat lungs

The objective of this study was to develop an X-ray computed tomographic method for measuring pulmonary arterial dimensions and locations within the intact rat lung. Lungs were removed from rats and their pulmonary arterial trees were filled with perfluorooctyl bromide to enhance X-ray absorbance. The lungs were rotated within the cone of the X-ray beam projected from a microfocal X-ray source onto an image intensifier, and 360 images were obtained at 1 degrees increments. The three-dimensional image volumes were reconstructed with isotropic resolution using a cone beam reconstruction algorithm. The vessel diameters were obtained by fitting a functional form to the image of the vessel circular cross section. The functional form was chosen to take into account the point spread function of the image acquisition and reconstruction system. The diameter measurements obtained over a range of vascular pressures were used to characterize the distensibility of the rat pulmonary arteries. The distensibility coefficient alpha [defined by D(P) = D(0)(1 + alphaP), where D(P) is the diameter at intravascular pressure (P)] was approximately 2.8% mmHg and independent of vessel diameter in the diameter range (about 100 to 2,000 mm) studied.     

High resolution electron tomography and segmentation‐by‐modeling interpretation in Bsoft

Bsoft offers many tools for the processing of tomographic tilt series and the interpretation of tomograms. Since I introduced tomography into Bsoft almost two decades ago, the field has advanced significantly, requiring refinement of old algorithms and development of new ones. The current direct detectors allow us to collect data more efficiently and with better quality, progressing towards automation. The goal is then to also automate alignment of tilt series and reconstruction. I added an estimation of the specimen thickness as well as fiducialless alignment, to augment the existing fiducial‐based alignment. High‐resolution work requires correction for the contrast transfer function, in tomography complicated by the tilted specimen. For this, I developed a method to generate a power spectrum using the whole micrograph, compensating for tilting. This is followed by routine determination of the contrast transfer function, and correction for it during reconstruction. The next steps involve interpretation of the tomogram, either by subtomogram averaging where possible, or by segmentation and modeling otherwise. Such interpretation actually constitutes the main time‐consuming part of tomography and is less amenable to automation compared to the initial reconstruction.     

TOM software toolbox: acquisition and analysis for electron tomography

Automated data acquisition procedures have changed the perspectives of electron tomography (ET) in a profound manner. Elaborate data acquisition schemes with autotuning functions minimize exposure of the specimen to the electron beam and sophisticated image analysis routines retrieve a maximum of information from noisy data sets. "TOM software toolbox" integrates established algorithms and new concepts tailored to the special needs of low dose ET. It provides a user-friendly unified platform for all processing steps: acquisition, alignment, reconstruction, and analysis. Designed as a collection of computational procedures it is a complete software solution within a highly flexible framework. TOM represents a new way of working with the electron microscope and can serve as the basis for future high-throughput applications.     

A protocol for Papanicolaou staining of cytologic specimens following flow analysis

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.     

A cone formation theory of contamination in high resolution transmission electron microscopy

The formation theory of the contamination cone in high resolution transmission electron microscopy is discussed on the basis of a time dependent series of micrographs and their tilted images for evaluating the contamination process. For a high current density of an electron probe focused on the specimen, it was observed that two contaminated cones covering both upper and lower surfaces of a carbon substrate are formed as a function of the exposure time of the electron beam. From the measurement of the cross-section and height of contaminated cones, the contamination rate together with the volume rate, and time dependent shapes of various cones were analysed. The analysis was dependent on the diffusion cross-section and current density together with an average lifetime of adsorption molecules.     

Automated electron microscope tomography using robust prediction of specimen movements

A new method was developed to acquire images automatically at a series of specimen tilts, as required for tomographic reconstruction. The method uses changes in specimen position at previous tilt angles to predict the position at the current tilt angle. Actual measurement of the position or focus is skipped if the statistical error of the prediction is low enough. This method allows a tilt series to be acquired rapidly when conditions are good but falls back toward the traditional approach of taking focusing and tracking images when necessary. The method has been implemented in a program, SerialEM, that provides an efficient environment for data acquisition. This program includes control of an energy filter as well as a low-dose imaging mode, in which tracking and focusing occur away from the area of interest. The program can automatically acquire a montage of overlapping frames, allowing tomography of areas larger than the field of the CCD camera. It also includes tools for navigating between specimen positions and finding regions of interest.     

Progress in applying the high-voltage electron microscope to biomedical research

This review attempts a physical definition of the technical problems and achievements in applying the high-voltage electron microscope (HVEM) to biological and medical research. It is hoped that the review will summarize for biologists, funding agencies, and institutions the achievements of the HVEM, its future prospects, and the main problem areas that still need to be explored. At present it is not known whether future HVEMs will favor the fixed beam or the scanning transmission electron microscopy (STEM) mode. The STEM mode offers reduced radiation damage as a result of more efficient electron detection and ease of manipulation of the collected signals by separating the elastic and inelastic signals. Energy filtration to remove the inelastic signal provides a means to enhance the contrast and improve the resolution for thick specimens. Several prototype STEM-mode HVEMs are now under development and it is expected that, in a few years, comparisons of fixed beam and STEM modes will be possible. The review discusses several HVEM instrument features that remain poorly developed. In the area of image recording a photographic emulsion has been designed to give optimized performance at an acceleration voltage of 1 MV. However, this remains unavailable commercially. Conversion of the HVEM electron image to a usable light image by phosphors etc., involves some difficulties, making it difficult to obtain good performance from TV systems. Since the HVEM is particularly useful for three-dimensional imaging, the further development of improved goniometers for stereo viewing and image reconstruction is important. The large volume available in the objective specimen volume and the increased penetration at high acceleration voltages make the HVEM particularly suitable for the application of environmental chambers in the microscopy and electron diffraction of thick wet specimens. An improved signal-to-noise ratio improves the prospects for elemental analysis at high acceleration voltages. When carefully carried out, improved resolution can be obtained in dark-field over that obtainable at 100 kV. Dark-field provides the easiest way to obtain high contrast on weakly stained or unstained objects. Its further improvement requires the use of specially thick and shaped beam stops and apertures that are not penetrated by the 1 MV beam. Recent HVEM studies of whole cells and microorganisms are reviewed. These studies already show that the former thin-section approach led to some incorrect ideas about the shape of some organelles and their three-dimensional relationships. This new information is proving important in helping to establish the function of fibrillar and membranous components of the cell. The most important limitation in examining thick sections is the large depth of field that causes excessive overlap of in-focus structures in stereo views of thick sections. In a few cases special specific heavy metal stains have been developed to overcome this problem, but an optical solution would be more generally applicable. Attempts are now being made to unscramble overlapped detail by applying the image reconstruction techniques of tomography and holography. It is concluded that even with existing techniques, the HVEM examination of thick sections provides a very useful improvement in sampling statistics and in three-dimensional imaging of cell structures over that obtainable by examining thin sections at a lower acceleration voltage (100 kV). Randomized author sequence.     

A dose-rate effect in single-particle electron microscopy

A low beam intensity, low electron dose imaging method has been developed for single-particle electron cryo-microscopy (cryo-EM). Experiments indicate that the new technique can reduce beam-induced specimen movement and secondary radiolytic effects, such as "bubbling". The improvement in image quality, especially for multiple-exposure data collection, will help single-particle cryo-EM to reach higher resolution.     

Effect of specimen size on work-of-fracture measurements

It has been suggested that work-of-fracture, which quantifies the ability of a material to resist fracture, is dependent on specimen size. This experiment compared work-of-fracture, calculated as energy per unit area, for different specimen sizes of Plexiglas, bovine tibial bone and aluminum. Three different geometrically similar cross sections were tested for each material for a total of 54 specimens. Work-of-fracture was measured by loading a notched beam (triangular cross section) in three-point bending at a constant deformation rate. The energy necessary to cause fracture was measured from a load-deformation curve. Specimen fracture area was determined using macrophotography. Atomic absorption spectrophotometry was used to determine weight percent calcium of bone specimens and quantitative light microscopy was used to determine fractional void area. Analysis of variance showed no effect of specimen size on work-of-fracture for aluminum or Plexiglas specimens (p greater than 0.05). A significant difference was found, however, between the large (area = 11.7 +/- 1.9 mm2) and small (area = 3.48 +/- 0.68 mm2) bone specimens and between the medium (area = 5.89 +/- 0.69 mm2) and small (area = 3.48 +/- 0.68 mm2) bone specimens. No correlation was found between work-of-fracture and either calcium content (r2 = 0.128) or fractional void area (r2 = 0.0713). The mean work-of-fracture values found are as follows: aluminum, 59.8 +/- 13.7 kJ m-2; Plexiglas, 0.620 +/- 0.074 kJ m-2; bone (area 5.89 +/- 0.69 mm2-11.7 +/- 1.9 mm2), 9.72 +/- 1.93 kJ m-2 and bone (area 3.48 +/- 0.68 mm2), 5.48 +/- 1.79 kJ m-2.     

Fabrication and Characterization of Disordered Polymer Optical Fibers for Transverse Anderson Localization of Light

We develop and characterize a disordered polymer optical fiber that uses transverse Anderson localization as a novel waveguiding mechanism. The developed polymer optical fiber is composed of 80,000 strands of poly (methyl methacrylate) (PMMA) and polystyrene (PS) that are randomly mixed and drawn into a square cross section optical fiber with a side width of 250 μm. Initially, each strand is 200 μm in diameter and 8-inches long. During the mixing process of the original fiber strands, the fibers cross over each other; however, a large draw ratio guarantees that the refractive index profile is invariant along the length of the fiber for several tens of centimeters. The large refractive index difference of 0.1 between the disordered sites results in a small localized beam radius that is comparable to the beam radius of conventional optical fibers. The input light is launched from a standard single mode optical fiber using the butt-coupling method and the near-field output beam from the disordered fiber is imaged using a 40X objective and a CCD camera. The output beam diameter agrees well with the expected results from the numerical simulations. The disordered optical fiber presented in this work is the first device-level implementation of 2D Anderson localization, and can potentially be used for image transport and short-haul optical communication systems.     

3D gold in situ labelling in the EM

We have developed a novel pre-embedding in situ hybridization labelling method for electron microscopy which has given much greater sensitivity and higher labelling levels than have been achieved previously, together with good ultrastructural preservation. Vibratome sections of plant tissue were labelled throughout their thickness with 1 nm gold antibodies and then silver enhanced, embedded in resin and sectioned for electron microscopy. Because the labelling extends throughout the depth of the specimen, this method permits the study of the 3D arrangement of the labelling at the electron microscope level by either stereo-pair recording, tomographic reconstruction or 3D reconstruction from serial sections. In this paper we describe the application of this method to study the organization of rDNA in pea root tissue.     

Identification of QTLs influencing agronomic traits in <Emphasis Type="Italic">Miscanthus sinensis</Emphasis> Anderss. I. Total height,flag-leaf height and stem diameter

We have developed the first quantitative trait locus (QTL) analyses for agronomic traits in a cross between F(1.1) (P1) and F(1.7) (P7) entries of Miscanthus sinensis Anderss. Both lines are offspring of the cross between MS-90-2 and MS-88-110. A map based on random amplified polymorphic DNA markers previously constructed was used to perform the QTL analyses. This map was developed using a new mapping strategy that has been designated offspring cross. Eleven QTLs were detected for height, panicle height and diameter using the programme mapqtl 4.0 and the multiple QTL method. QTL significance was determined using several analyses, including Kruskal-Wallis analyses, empirical determination of LOD critical values using permutation tests, QTLs validation with field data over 2 years and co-localization of QTLs for correlated traits. The results obtained could be the first step in developing a marker-assisted selection programming in this species for biomass production.     

Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.