Three-dimensional reconstruction of microtubule-containing structures from electron micrographs

Microtubules and associated structures are among the more difficult samples studied by 3D reconstruction techniques because of their size, complexity, and lability. Nevertheless, their importance in many cellular functions often justifies efforts to acquire 3D information up to the resolution limit of negatively stained specimens or beyond. A combined approach which utilizes methods appropriate to both helical symmetry and 2D crystal lattices seems to provide the surest route to 3D reconstruction. Images of unstained specimens obtained by newer techniques of microscopy present challenging problems in data analysis, but potentially offer higher-resolution information.     

A comparison of different intensity gradient formulae for orientation analysis of electron micrographs

A detailed study of different intensity gradient formulae was conducted using 91 separate images covering a wide range of microfabric. It was found that a formula based on the 3 × 3 local pixel array gives almost identical results over all images with the standard formula based on 20 pixels forming a circular grouping within the 5 × 5 local array. Other formulae based on other groupings of pixels within the same pixel arrays give markedly different results. Computation times are reduced up to 45% when the 8,5 formula is used in place of the 20,14 one, and would thus seem to have advantages when large numbers of images are to be processed. While all formulae give somewhat similar domain-segmented images when viewed visually, differences are noted both in the number of segmented domains as well as in the area of domains in each orientation sector. It is possible that the radius of the modal filter selected for the domain segmentation should be varied according to the formula type used.     

Image registration in electron microscopy: Application of a robust method

The geometric registration of two electron microscopic images generally is performed by maximizing the cross-correlation coefficient between them. We show that a new similarity measure (the number of sign changes) is useful for performing simultaneously geometric and gray-level registration. This method is robust, which means that it provides a good estimation of the parameters even in the presence of outliers that cannot be described by the registration model.     

A comparison of three different electron microscopical grade glutaraldehydes used to fix ovarian tissue

‘EM grade’ glutaraldehydes from three suppliers were tested for the following: (1) pH of stock solution, (2) 235/280 nm UV absorbance ratio, (3) milliosmolarity of 2% and 4% solutions, (4) quality of fixation produced in ovarian tissue by fixatives prepared from each stock solution. Low pH of the stock solution appeared to be the most detrimental factor in fixation. Fixatives with a low 235/280 nm ratio (between 0·18 and 0·67) did not give good fixation regardless of the pH of the stock solution. Fixatives made from stock solutions with a pH above 4 and a 235/280 nm ratio between 1 and 2 resulted in the best preservation of ultra-structure. Variations in milliosmolarity from one batch of stock solution to another were encountered, but appeared to have no correlation with the quality of fixation.; Received 1 October 1973; revision received 20 December 1973     

Crystallographic analysis of freeze-fracture electron micrographs: application to the structure determination of cubic lipid–water phases

An original approach combining freeze-fracture electron microscopy and quantitative image processing has been developed as an alternative to X-ray analysis. It has been applied to the crystallographic study of different lipid–water cubic phases [bicontinuous or micellar and of type I (oil-in-water) or type II (water-in-oil)] and has enabled significant advances in the study of these phases. Freeze-fracture electron microscopy has revealed that the cubic phases fracture preferentially along a few crystallographic directions which appeared on the images as noisy planar fracture surfaces containing periodic information. The visibility of the corresponding unit cells has been considerably improved by image-filtering techniques based on correlation averaging, allowing a quantitative analysis of the fracture images to be made. This analysis yielded faithful information on the symmetries of the cubic structure (rotation axes and mirror planes) as well as on the structure of the cubic phase itself. Eventually, the different parameters that determine the most favourable fracture pathways within the structures were established. This novel approach constitutes a powerful tool of general interest, complementary to X-ray diffraction, for solving complex ordered macromolecular structures at low resolution.     

The use of histograms for transmission electron microscopy: Defocus and astigmatism correction at high resolution

A real-time method for optimizing the defocus of a conventional transmission electron microscope in the phase contrast imaging mode has been investigated using image histogram data. This method can also be used to minimize the objective lens astigmatism. It will be shown both theoretically and empirically, using a digital television frame store, that a histogram will give the largest peak when an image has a broad and flat contrast transfer function. This method has distinct advantages of speed and minimal computational requirements over obtaining the power spectrum of an image.     

Progress in electron microscopic diagnostics: semi-quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea-pig

Potassium antimonate was used to precipitate calcium in the cochlea of the guinea-pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10-μm² area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi-quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca²⁺ -edge signals, the combined signal of the antimony M_4,5-edge and the oxygen K-edge of the calcium antimonate salt formed was chosen for the semi-quantitative estimation. Images of the inelastically scattered electrons of the precipitates at ΔE = 570 eV were recorded by electron spectroscopic imaging. The area covered by the calcium precipitates within a given cell type was determined in different ultrathin sections of the same organ of Corti by an image processing system.     

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: The utility of methylamine tungstate stain and butvar support film in the study of macromolecules by transmission electron microscopy

The structure of ornithine decarboxylase (M_r ≈? 1.04 × 10⁶) from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted ± 45°. The two edge views were related by a 30° rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-Å resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10° with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.     

A comparison of the relative friction and wear responses of PTFE and a PTFE-based composite when tested using three different types of sliding wear machines

In this work, the tribological behavior of a commercially available polytetrafluoroethylene-based composite material filled with polyimide microparticles has been thoroughly investigated using different approaches. Specifically, two standard tribological tests, i.e., pin-on-disc and thrust washer measurements, have been compared with sliding tests performed on real components using a specially designed lab bench. The obtained results demonstrated that, despite the different testing methods (continuous rotation vs. reciprocating linear movement for the pin-on-disc/thrust washer and sliding tests, respectively), the different techniques provide highly comparable data within the adopted experimental conditions and can be successfully combined to assess the overall tribological features of this PTFE-based polymer composite.     

Imaging properties of bright-field and annular-dark-field scanning confocal electron microscopy: II. point spread function analysis

The imaging properties of bright field and annular dark field scanning confocal electron microscopy (BF-SCEM and ADF-SCEM) are discussed based on their point spread functions (PSFs) in comparison with multislice simulations. Although the PSFs of BF-SCEM and ADF-SCEM show similar hourglass shapes, their numerical distributions are quite different: BF-SCEM PSF is always positive and shows a center of symmetry whereas the ADF-SCEM PSF is complex and has Hermitian symmetry. These PSF properties explain the large elongation effect in BF-SCEM for laterally extended object and almost no-elongation in ADF-SCEM, illustrating the importance of the numerical analysis of PSFs. The Hermitian symmetry of the ADF-SCEM PSF results in an interesting “edge enhancement effect” at the interface. Simulation using the PSF and the multislice method verified this effect at GaAs surfaces and InAs interfaces embedded in GaAs. This unique feature of ADF-SCEM can potentially be useful for depth sectioning. It is also pointed out that a PSF imaging model cannot be applicable for BF-SCEM of a phase object, when the system is symmetric and aberration free.     

Water flow measurement in large bore pipes: an experimental comparison between two different types of insertion flowmeters

In this paper the metrological behavior of two different insertion flowmeters (magnetic and turbine types) in large water pipes is described. A master-slave calibration was carried out in order to estimate the overall uncertainty of the tested meters. The experimental results show that (i) the magnetic insertion tested flowmeter performs the claimed accuracy (+/- 2%) within all the flow range (20:1); (ii) the insertion turbine tested meter, instead, reaches the claimed accuracy just in the upper zone of the flow range.     

Minima equalization: A useful strategy in automatic processing of microscopic images

Specifically stained features in microscopic images rarely have a unique range of grey levels which would permit selection of the features by simple thresholding. The spaces between features and immediately surrounding them are often as dark or darker than the more lightly stained features. An algorithm for minima equalization which facilitates the extraction and segmentation of such features was designed and is explained in intuitive terms. It is applied to the analysis of cross-sections of peripheral myelinated nerve fibres. It is shown that the binary image obtained can be combined with a gradient image to give a binary image which accurately reflects the thickness of the myelin in the original image. Using silver-impregnated nerve endings and bile canaliculi stained for thiamine pyrophosphatase, binary images like those prepared manually from thick specimens using a camera lucida can be obtained using the minima equalization procedure. The image processor is used to develop a composite image by combining images at various focal planes through the thick specimen. This image is then processed to obtain the binary image.     

A quantitative approach to cytoarchitectonics: Analysis of structural inhomogeneities in nervous tissue using an image analyser

Cytoarchitectonic investigations are based on the analysis of structural inhomogeneities in the neuronal composition of nervous tissue. Boundaries of brain regions are established at locations where local structural properties, such as density, shape, orientation or arrangement of the nerve cells, change. A quantitative approach requires a complete scanning of histological sections and the measurement of at least one of these properties using an automatic device. In contrast to recently developed stereological methods which result in unbiased estimates of parameters and which are based on efficient sampling procedures, measurements with automatic devices are biased due to section thickness and problems in the segmentation of nerve cell bodies. Automatic measurements are necessary, however, if a complete scanning of histological sections is required in order to localize areal or laminar boundaries. In this approach, the grey level index (GLI) is measured with a TV-based image analysing system from routine histological sections. Using Nissl-staining, this parameter is a biased estimate of the local volume density of Nissl-positive structures (cell bodies). The histological section is digitized into a GLI image by a scanning procedure. The GLI image is processed by image enhancement procedures in order to visualize the laminar pattern. Areal boundaries are localized at positions where this laminar pattern changes. GLI statistics of single brain regions can easily be evaluated by delineating these regions with a cursor in the GLI images of the sections. Information from a series of sections is compiled by specific application programs.     

Dynamic compensation of load cell response: A time-varying approach

This paper presents a new method for dynamic compensation of the load cell response using linear time-varying continuous-time filter. Load cells have an oscillatory response which always needs time to settle down. Therefore, it is justified to search a technique for an effective improvement of the sensor response. The paper describes a theoretical implementation of the proposed time-varying filter, and suggests the implementation technique with the aid of which this kind of filter can be implemented in practice. Simulation results verifying the effectiveness of the proposed filter are presented and compared to the traditional time-invariant configuration.     

Processing of X-ray tomographic images: A procedure adapted for the analysis of phase distribution in MellapakPlus 752.Y and Katapak-SP packings

We developed a practical methodology to face common problems encountered in the analysis of tomographic images to get quantitative information on phase distributions in gas liquid contacting columns packed with complex metallic packings. In the present study, the procedure was developed and validated for MellapakPlus 752.Y and Katapak-SP packings, but it may be applied to any type of metallic or modular packing of similar dimensions. Steps of tomographic image processing and associated parameters (threshold, calibration factors for solid and liquid phase) were first determined on images of dry and flooded packing elements of MellapakPlus 752.Y. The parameter values were then adapted to analyse images of dry and flooded packing elements of Katapak-SP, which are made of an alternation of MellapakPlus 752.Y sheets and catalytic baskets. In a certain way, MellapakPlus 752.Y and Katapak-SP (11 and 12), the global geometrical properties of which are well known, are used as physical phantoms to develop a validated image post-processing procedure.The latter is used to quantitatively analyse liquid distribution images in irrigated MellapakPlus 752.Y and Katapak-SP (11 and 12) packings, giving access to spatial distribution of local values of hydrodynamic quantities such as the liquid holdup and gas-liquid interfacial area. Global values of the liquid hold-up, relative to the whole packed bed volume are determined by averaging tomographic results over the column height. They are successfully compared with bed scale data measured on the same MellapakPlus 752.Y and Katapak-SP (11 and 12). This analysis further validates the procedure used for the processing of tomographic irrigated images.So the proposed methodology enables one to obtain quantitative information on the spatial distributions of structural quantities as well as of hydrodynamic quantities in complex metallic packings, which are fully validated on the basis of a comparison with global values obtained by well-established measurement techniques.     

Qualitative analysis of smear layer treated with different etidronate concentrations: A scanning electron microscopy study

The aim of this study was to examine the smear layer removal capacity of various etidronate treatments. Seventy‐eight roots were instrumented up to apical size X4 and randomly divided into five treatment groups and a positive control group (n = 13). Groups were as follows: irrigation with 5 ml of sterile saline (control) for 3 min, 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) for 3 min, 5 ml of 9% A1‐hydroxyethylidene‐1,1‐bisphosphonate (HEBP) for 3 min, 5 ml of 18% HEBP for 3 min, irrigation with 5 ml of 1% sodium hypochlorite (NaOCl) + 9% HEBP for 3 min, and 5 ml of 2% NaOCl + 18% HEBP for 3 min. The roots were examined using a scanning electron microscope. The smear layer scores in the 2% NaOCl + 18% HEBP group were lower than those in the other treatment groups and the control group (p < .05). There were no significant differences among the treatment groups in the smear layer scores of the coronal and middle thirds. However, the smear layer scores in the 2% NaOCl + 18% HEBP treatment group were lower those in the 9% HEBP and 18% HEBP treatment groups in the apical third (p < .05). In the clinical setting, the recommended concentration of HEBP is 18%, and it should be used with an oxidizing agent to ensure optimum smear layer removal.     

Relationship between the structure of C60 and its lubricity: A review

Based on analyses of the calculations of quantum theories, such as electron charge density, the energy level distribution of orbitals, and the forces between C atoms and between the molecules in C₆₀ crystals, the author concludes that C₆₀ crystal is a promising solid lubricant. Furthermore, owing to the unique spherical structure of its molecules, high chemical stability, and the phase transition of the crystal fullerene C₆₀ may have some particular tribological properties that are superior to traditional graphite and MoS₂, especially under crucial conditions such as high vacuum, high pressure or in a humid environment. The tribological tests that have been reported show the lubricity of fullerene C₆₀, and the effects of the lubricating properties of C₆₀ on some metals are also predicted.     

A method for structure analysis of nanomaterials by electron diffraction: Phase identification and unit cell determination

We report a quick and easy method for a random selected area electron diffraction (SAED) pattern without rotating and tilting the specimen to perform phase identification and unit cell determination by combined with the XRD softwares. If your TEM is well aligned and camera length is carefully corrected, two‐dimensional (2D)‐SAED pattern can be directly transformed to 1D‐profile after the center determination of pattern, this profile is then imported to XRD analysis packages. Finally, phase identification and unit cell determination can be performed after peak search or precise peak position determined by profile fitting. Two examples, flaky‐like TiO₂ nanomaterial and TiO₂ nanotubes precipitated by the silver nanoparticles, were tested and verified for the validation of phase identification and unit cell determination using this method; the successful crystallographic analysis of one single gold nanocrystal indicates it is still validate for the nanocrystals with the smaller diffraction volume, but need two or more random tilt SAED patterns. This method could be further used in the quantitative phase analysis, structure determination and Rietveld refinement for the nanomaterials if the reliable integrated intensity can be extracted. Microsc. Res. Tech. 76:641–647, 2013. © 2013 Wiley Periodicals, Inc.