Pronounced loss of cell nuclei and anisotropic deformation of thick sections

The local deformation and variations in section thickness are studied in 100-μm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 μm and the lower 10 μm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.     

Electron microscopy of semithick sections: Advantages for biomedical research

Many transmission electron microscopes are available which can be used to examine biological material in 0.25–0.50-μm-thick sections. When compared to the traditional thin section, these “semithick” sections possess a number of inherent advantages: They can be screened for content with the phase contrast light microscope, they facilitate many types of studies requiring an analysis of serial sections, and they are frequently the optimum thickness for stereomicroscopy. Structures such as microtubule-associated components, as well as structural relationships between cellular constituents, may also be clearly visible in semithick sections which are not visible, or go unnoticed, in thin sections. Together these advantages enable an investigator to obtain a more complete three-dimensional picture of a cell or cell component in a significantly (i.e., up to 90%) shorter period of time than would be required if thin sections were used. Semithick sections may, therefore, make a study feasible which is not approachable, or which is approachable only with great difficulty, by conventional thin sectioning techniques.     

Is there section deformation resulting in differential change of nuclear numerical densities along the z axis of thick methacrylate or paraffin sections?

The optical disector, a three‐dimensional counting frame or probe in stereology, is often positioned in the middle (depth) of a thick section for unbiased nuclear counting. Using 30–40 μm thick methacrylate or paraffin sections for nuclear counting of neurons with the optical disector, however, some studies showed markedly higher nuclear densities at 10% of the section thickness near the top or bottom surface of the section, suggestive of deformation of section along its z axis and thus affecting the number estimation. To verify the findings, this study obtained two sets of 12–14 methacrylate sections (average thicknesses 21.7 and 29.4 μm) and two sets of 12 paraffin sections (average thicknesses 13.8 and 29.2 μm) from mature rat testes. Each section was used to count round spermatid nuclei in the seminiferous epithelium densely packed with the cells, using 3–4 consecutive disectors placed vertically (along the z axis of the section) from the top surface of the section, through the whole section thickness (two sets of methacrylate and paraffin sections) or in 80–83% of the thickness (other sections). The results demonstrated that, overall, there were no considerable nonuniform changes of the nuclear densities along the z axis of the sections.     

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.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

SLS烧结参数对原型精度影响的正交试验分析

通过对轴对称圆柱孔等典型几何形状的烧结成型,研究了SLS中激光功率、加热温度以及切片厚度这三个烧结参数对圆柱孔形位公差以及直径和圆柱度的误差的影响。采用正交试验方法,在两种不同的切片方式下,分析了各种误差产生的主要原因,以及获得较高原型精度的工艺参数值。     

Can you trust your cryostat? Reproducibility of cryostat section thickness

Reproducibility of cryostat section thickness is required for valid quantitative microscopy. This is generally pursued by motorized sectioning using a low but constant speed. The purpose of our study was to compare variation in section thickness between motorized and manual cryostat sectioning. Serial sections were cut from a frozen block of homogenized tissue on different days. Lactate dehydrogenase activity was histochemically detected and calibrated absorbance measurements were taken. The coefficients of variation of measurements was 9.7% for motorized sectioning and 3.3% for manual sectioning. In conclusion, section thickness is similarly reproducible after manual sectioning compared with motorized sectioning, if not better.     

Estimation of the average section thickness in ribbons of ultra-thin sections

A simple procedure for estimating the depth to which a ribbon of ultra-thin sections cuts into a block is described. The average section increment is obtained by dividing the depth by the number of sections in the ribbon. Comparison with measurements of individual sections made using interference microscopy suggests: (a) that loss of resin brought about by the cutting process is insignificant, i.e. that the average section increment equals the average section thickness, and (b) that there can be considerable variation in section thickness along a ribbon. Provided that such variation is taken into account, the method based on measurements of the block face is simple, avoids time-consuming alternative procedures, and is appropriate when seeking to determine the depth dimension to be used in making three-dimensional reconstructions from long sequences of sections.     

An improved online dimensional measurement method of large hot cylindrical forging

The dimensional measurement of large forgings under high temperature plays an important role in product control. Cylindrical forgings belong to the important products in the forging workshop. In this paper, an improved online measurement method based on binocular vision for the dimensions of hot cylindrical forgings is proposed. Firstly, images of hot cylindrical forgings are captured by two CCD cameras, and the distorted light stripes projected onto hot cylindrical forgings can be resolved from the image by the deviated stripe model. Then, an accurate sub-pixel extraction algorithm of center points of the light stripes is developed for making the extraction of center points of the lights stripes more accurate. Moreover, for changing the field of view of cameras to measure different sections of forgings in the process of manufacturing, the fundamental matrix F is automatically calculated by matching the intersecting points of the projected light stripes in different images, without interrupting the forging process. Finally, experiments on measuring the diameters of hot cylindrical forgings in the workshop are conducted, and the experimental results indicate that the proposed measurement method is effective. Besides, an additional experiment in the laboratory verifies that the relative error of measuring diameters by the presented method is less than 0.7%.     

Thickness estimation of fluorescent sections using a CSLM

A novel method for the measurement of the section thickness of LM-plastic embedded specimen has been developed. This method makes use of the fluorescence that comes from standard routine stainings such as haematoxylin and eosin and periodic acid-Schiff. An x-z profile of the specimen is first scanned using a confocal scanning laser microscope. The full-width half-maximum, FWHM, of the profile is computed and used as a measure of the specimen thickness. This method has proven to be simple and quick: a slide with five sections takes less than 1 min to measure. A theoretical treatment is presented which shows that the FWHM of the axial fluorescent profile is a good estimate of the actual thickness when the sample thickness is greater than 20 (when thickness is expressed in generalized longitudinal optical coordinates). This corresponds to a thickness of about 1μm when using an NA=1.3 oil-immersion objective and 488-nm excitation. The relative error in thickness is then less than 10%. Simulations and experiments have been carried out to examine how problems such as bleaching, sample tilt and curvature of field influence the FWHM. The results show that the method is robust and insensitive to such problems.     

Reconstruct: a free editor for serial section microscopy

Many microscopy studies require reconstruction from serial sections, a method of analysis that is sometimes difficult and time-consuming. When each section is cut, mounted and imaged separately, section images must be montaged and realigned to accurately analyse and visualize the three-dimensional (3D) structure. Reconstruct is a free editor designed to facilitate montaging, alignment, analysis and visualization of serial sections. The methods used by Reconstruct for organizing, transforming and displaying data enable the analysis of series with large numbers of sections and images over a large range of magnifications by making efficient use of computer memory. Alignments can correct for some types of non-linear deformations, including cracks and folds, as often encountered in serial electron microscopy. A large number of different structures can be easily traced and placed together in a single 3D scene that can be animated or saved. As a flexible editor, Reconstruct can reduce the time and resources expended for serial section studies and allows a larger tissue volume to be analysed more quickly.     

The ‘corpuscle’ stereological problem—re-evaluation using slab fragment volumes and application to the correction of DNA histograms from sections of spherical nuclei

A new category of stereological size distribution unfolding models is introduced. It is based on the use of the volumes of particle slab fragments, in addition to their profile dimensions. When spheres are cut by a slab of known (constant) thickness, an estimation of discrete sphere sizes from section data is then possible, as only one parent sphere solution exists for any slab fragment, given the latter's projection size and volume. The unfolding algorithm consists in sequentially testing a set of equations: only one of the solutions satisfies various constraints on bounds. A precise determination of the section thickness is required. Truncation parameters, instead of being troublesome inputs as in classical unfolding models, become valuable outputs. This model offers the first stereologically valid solution to the important problem of correcting DNA-amount histograms obtained from sectioned spherical nuclei. Under the (biologically reasonable) assumption that the nuclear volume is proportional to the DNA amount, it is possible to estimate the DNA concentration and, subsequently, compute discrete slab fragment volumes from corresponding DNA values. An application to Feulgen-stained rat liver sections is shown. Measurements of hepatocytic nuclear-profile areas and integrated optical densities are obtained by automated image analysis (IBAS), and the model is used to unfold the section-obtained DNA histogram. A maximum likelihood fitting of the final distribution with chi functions allows a parametric estimation of ploidy frequencies. This model can only be used for acceptably spherically shaped particles.     

Design-based estimation of surface area in thick tissue sections of arbitrary orientation using virtual cycloids

Surface area is a first‐order stereological parameter with important biological applications, particularly at the intersection of biological phases. To deal with the inherent anisotropy of biological surfaces, state‐of‐the‐art design‐based methods require tissue rotation around at least one axis prior to sectioning. This paper describes the use of virtual cycloids for surface area estimation of objects and regions in thick, transparent tissue sections cut at any arbitrary (convenient) orientation. Based on the vertical section approach of Baddeley et al., the present approach specifies the vertical axis as the direction of sectioning (i.e. the direction perpendicular to the tissue section), and applies computer‐generated cycloids (virtual cycloids) with their minor axis parallel to the vertical axis. The number of surface‐cycloid intersections counted on focal planes scanned through the z‐axis is proportional to the surface area of interest in the tissue, with no further assumptions about size, shape or orientation. Optimal efficiency at each x–y location can be achieved by three virtual cycloids orientated with their major axes (which are parallel to the observation planes) mutually at an angle of 120°. The major practical advantage of the present approach is that estimates of total surface area (S) and surface density (S_V) can be obtained in tissue sections cut at any convenient orientation through the reference space.     

Vitreous cryo-sectioning of cells facilitated by a micromanipulator

Sectioning vitrified cells and tissues for cryo‐electron microscopy is more challenging than room‐temperature sectioning of plastic‐embedded samples. As the sample must be kept very cold (相似文献   

压杆稳定设计的直接迭代法

研究了一种压杆稳定设计的迭代法——直接迭代法,利用Maple编写了压杆设计迭代法的计算机程序。对迭代法程序进行了系列算例设计。用直接迭代法设计了连杆的圆截面直径:对于给定不同的柔度初始值,其结果在各柔度范围内都是收敛的。对不同给定参数(杆长,支承,轴向压力),系列设计了截面直径。实例表明:直接迭代法适合截面是规则图形的压杆稳定性设计。迭代法与Maple语言相结合设计压杆,稳定性好,精确性高,可靠性强,快速、便捷,大大提高了计算效率。所编的程序可进一步扩充,扩大其应用范围。     

Low-energy electron/atom elastic scattering cross sections from 0.1–30 keV

Empirical forms for electron/atom scattering cross sections predict backscattering factors that compare well with those calculated using tabulated Mott data from 0.1 to 30 keV. The form of the empirical total cross section is similar to the screened Rutherford cross section. The fit to the tabulated differential Mott cross sections is decomposed into two parts, one part being of the same mathematical form as the screened Rutherford cross section (σ_R), and the second part being an isotropic distribution (σ_I). The ratio of the total cross sections (σ_R/σ_I) between the screened Rutherford part of the differential scattering cross section and the isotropic part of the distribution is fitted to give the same ratio of forward to backscattered currents as the tabulated Mott differential cross sections. The three equations, one for the total elastic cross section and two describing the differential cross section—one for the Rutherford screening parameter and one for the ratio σ_R/σ_I—give backscattering results covering all the major trends with energy and atomic number compared with the backscattering coefficients calculated using tabulated Mott cross sections. However, agreement with experiment is poor for some well-researched examples such as Au. Monte Carlo calculations using the empirical cross sections show that surface effects may be critical in interpreting experimental results.     

A method for evaluation of elliptical profiles

Measurement and evaluation of circular profiles of cylindrical components in terms of circularity has been well established using roundness measuring instruments. For elliptical sections having very little difference between the major and minor diameters this difference can be measured with the roudness measuring instrument. However, fitting of the best fit ellipse and hence the definition of ellipticity are not available. Applications of elliptical profiles include instruments, such as the photospectrometer developed at CMTI with accurate elliptical cams, elliptical gear blanks, and the pistons of internal combustion engines whose cross sections are elliptical. In this paper a method for the evaluation of the elliptical profiles for centre and orientation of major and minor axes is described and two concepts for defining ellipticity are discussed     

Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope

A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 μm thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.     

Direct determination of ultrathin section thickness using latex particles

A method is proposed to determine ultrathin section thickness by means of noncentral sections obtained from latex spheres. The resulting spherical segments provide the parameters necessary to calculate very simply the section thickness without further treatment. The method is illustrated by an example using the grid-sectioning technique and could be generalized to classical microtomy.     

The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique

The thickness of ultrathin tissue sections embedded in Epon-Araldite and cut with a diamond knife was measured by re-sectioning and electron microscopic examination of the section profiles. A secondary section mounted on a Formvar-coated slot grid provided enough normally cut segments (seven to seventeen) for measurements giving a precise estimate of mean thickness, comparable to that obtainable by interference microscopy (±2.3% or less for grey to dark gold sections). The standard deviation of section thickness within sections was never more than 5 nm, corresponding to a coefficient of variation of 6.5% or less for sections more than 48 nm thick. This suggests that variation in section thickness, within sections, may be less than has been supposed, so that quantitative work may be based on thickness measurements made over a limited representative area. A silver interference colour was associated with sections 49–60 nm thick.