Scanning electron microscopy,autolysis, and irradiation as techniques for studying small intestinal morphology

Examination of autolysed control mouse small intestine using scanning electron microscopy has revealed details of the connective tissue components of the mucosa. The cores of the villi are seen collapsed across the intervillous basin. Crypts of Lieberkuhn are seen as tubular channels stretching down from the intervillous basin. Sometimes the crypts are split in two by a connective tissue septum. The mouths of the crypts of Lieberkuhn are, in general, arranged in double rows between the single rows of villi. The ratio of number of crypts to numbers of villi was calculated as 5.01:1. This is close to the figure of 4.53:1, as quoted by Smith & Jarvis (1980) who used differential interference contrast microscopy to investigate the crypt to villus ratio. After radiation, the severe drop in the number of crypt mouths can be clearly seen by the combination of autolysis and scanning electron microscopy: the rows of crypt mouths between the villi have been lost, and many crypt mouths have been occluded by stromal tissue. The arrangement of the crypt mouths and the observation of mucosal abnormalities after irradiation have led to the postulation that cells leaving the crypt mouths move in a spiral manner towards and then up the villous surface: this postulated movement might imply an asymmetry in some properties of enterocytes. The use of scanning electron microscopy in conjugation with autolysis and irradiation has thus forced a critical re-examination of the relationships between crypts and villi.     

Investigation of dislocation geometries in the diamond cubic structure

The weak-beam technique of electron microscopy (Cockayne, Ray & Whelan, 1969) has been applied to the examination of dislocations in germanium. These are shown to be dissociated into partial dislocations with a separation in the edge orientation of 5.5 ± 1·0 nm. A value for the stacking-fault energy of γ = 60 ± 8 mJ m^?2 (erg cm^?2) is deduced from the measured dissociation width as a function of orientation, using anisotropic elasticity theory.     

Dimensions of active cytochrome c oxidase in reconstituted liposomes using a gold ball shadow width standard: a freeze-etch electron microscopy study

The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported. Freeze-etched cytochrome oxidase vesicles and gold ball calibration grids were simultaneously shadowed with platinum/carbon. The shadow width distribution of the cytochrome oxidase located in and spanning the membrane was measured. The membrane fracture face edge and cross-fractured bilayer membrane edge were also measured. Dimensions of the cytochrome oxidase were found to be 5·8 ± 0·3 nm in diameter parallel to the membrane and 8·2 ± 0·3 nm long across the membrane. The bilayer membrane dimensions were 3·0 ± 0·3 nm for the half bilayer and 5·8 ± 0·3 nm for the cross-fractured bilayer membrane edge thickness. The length of the cytochrome oxidase was observed to span the bilayer membrane. Previous X-ray diffraction measurements on similar hydrated liquid crystalline artificial membranes were found to be in good agreement with the freeze-etched results. Membrane widths from thin-sectioned cytochrome oxidase vesicles were measured and found to be 5·8–5·9 nm in non-post-stained sections. Post-staining with uranyl acetate and/or lead citrate was shown to increase this average thickness. The technique of freeze-etching electron microscopy in conjunction with the gold ball shadow width calibration experiment has been shown to provide accurate and precise measurements of membranes and a functional intramembrane protein in a hydrated non-crystalline sample.     

Quantitative analysis of variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) of cell/substrate contacts

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm²) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm²).     

Optimization of STM/FIM nanotip aspect ratio based on the Taguchi method

This paper presents optimization of electrochemical etching parameters to achieve the optimum aspect ratio of the scanning tunneling microscopy/field ion microscopy tungsten nanotip by using Taguchi method. The combination of optimum level of process parameters was obtained by using the analysis of signal-to-noise ratio. The level of importance of the process parameters on the nanotip aspect ratio was determined by using analysis of variance. It was found that the optimum level of process parameters are electrolyte concentration of 2 M/lit, wire immersion length of 2.5 mm, cathode tube inner diameter of 40 mm, and voltage of 3.5 V. Within the range of experiments and the process parameters in terms of impact significance were found to be electrolyte concentration, process voltage, wire immersion length, and inner diameter of cathode tube, respectively. By using the optimum level of the process parameters, the nanotip aspect ratio was enhanced by 263% in comparison to the mean value of the experimental results. The nanotip aspect ratio of up to 163:1 was obtained in the present research.     

Study of solid particle erosion on AISI D2 using angular silicon carbide and steel round grit particles

Abstract

In this study, the performance of AISI D2 steel subjected to solid particle erosion tests was analysed. This material has applications for tools and dies for blanking, wood milling cutters, cold-extruding and other operations requiring high compressive strength and excellent wear resistance. The erosion tests performed by using a rig developed according to some parameters of the ASTM G76-95 standard. Two abrasive were used, angular silicon carbide (SiC) and steel round grit, both, with a particle size of 400–420 μm. This allowed comparing the erosion severity of each abrasive particle. The tests were conducted using four different incident angles 30, 45, 60 and 90° with a particle velocity of 24±2 m s^?1 and a flow rate of 21±2·5 g min^?1 for silicon carbide and 48·5±3·5 g min^?1 for the steel round grit. The exposure testing time was 10 min. Subsequently, the surface damage was analysed with a scanning electron microscope (SEM) to identify the wear mechanisms. Additionally, atomic force microscopy (AFM) was conducted in order to obtain roughness of the surface damage at 60°. The results indicated that higher amount of mass loss was obtained by angular silicon carbide particles.     

Tem Of Dislocations Under High Stress In Germanium And Doped Silicon

The stacking fault energies y of silicon (58 ± 6 mJ m^?2) and germanium (75 ± 10 mJ m^?2) were determined. Within the limits of accuracy γ was not found to change on doping with (13·8 mol m^?3 (8 × 10¹⁸ cm^?3) boron, and 1·17 mol m^?3 (7 × 10¹⁷ cm^?3) phosphorus). Freezing in dislocations under high shear stress reveals a different behaviour of screw dislocations: whereas these dislocations become wider in pure and p-silicon, they become narrower in n-silicon. From this we conclude the ratio of mobilities of the two 30° partials to be different in n- and p-silicon. Other observations on frozen dislocations are mentioned.     

Quantitative water mapping of cryosectioned cells by electron energy-loss spectroscopy

A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 10³ e/nm² limits the useful spatial resolution to around 80 nm for ± 5% precision at a single pixel. By averaging pixel intensities a value of 56·8% with a precision of ± 2·0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.     

Morphological,microscopic, and physicochemical studies of Diospyros montana

Adulteration is the root cause of producing not only a chemically and pharmacologically inferior but also in some instances hazardous or poisonous drug. Despite availability of several techniques, microscopy and physicochemical analyses are the most practical approaches for crude drug authentication. Hence, the present study aimed to evaluate morphological, microscopic, and physicochemical properties of root, bark, leaf, and fruit of Diospyros montana Morphological properties were determined by sensory organs, whereas microscopic features of cross‐sections and powders were determined by light and scanning electron microscopy. The proximate and fluorescence analyses were performed using the standard guidelines. The physical examination of fresh, shade‐dried, and powdered material showed no significant change in color. The identifying cellular structures included cuboidal cork, pitted tracheids, scalariform, reticulate and spiral xylary vessels, and rosettes, raphide, and cuboidal calcium oxalate crystals. The stomatal number, stomatal index, vein‐islet and vein‐termination number, and palisade ratio in the leaf were found to be 293.91 ± 32.68 mm^?2, 64.18 ± 3.42%, 22.00 ± 3.81 mm^?2 and 38.40 ± 5.81 mm^?2, and 3.85 ± 0.60, respectively. Total ash, acid insoluble ash, water soluble ash and sulfated ash of leaf (9.00 ± 0.50%, 1.67 ± 0.23%, 2.00 ± 0.22% and 14.50 ± 0.99%, respectively), foaming index of bark and root (111.11 ± 2.11), and swelling index of fruit (19.00 ± 3.45) were higher than the other parts. The powder of different parts showed characteristic colors in the daylight and UV light upon treatment with various regents. The plant was found to be rich in saponins, fibers, and flavonoids. The results of the present study may serve as identifiers of different parts of Diospyros montana.     

Morphometric,quantitative, and three‐dimensional analysis of the heart muscle fibers of old rats: Transmission electron microscopy and high‐resolution scanning electron microscopy methods

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high‐resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A‐, I‐, and H‐bands and their M‐ and Z‐lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm², a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

The effect of refractive index of the mounting medium on the apparent diameter of latex particles and glass fibres

The diameters of latex particles were measured in different media with a light microscope. The microscope was fitted with a filar micrometer eyepiece and, for measurement, the cross-wire was set on the centre of the dark line forming the edge of the image. Electron microscopy was used for comparison. Good agreement between light and electron microscopy was obtained using media of relative refractive index (n_d object/n_d medium) between 1·06 and 1·08. With higher relative refractive indices the light microscope underestimates dimensions for particles 4·17 μ diameter and overestimates for particles 2·7 μ diameter.     

An electron diffraction study of paramylon storage granules from Euglena gracilis

Paramylon storage granules from Euglena gracilis were characterized by electron diffraction techniques using electrons of various accelerating voltages: 2 MV for the thick granules and 100 kV for the thinner ones. Intact granules gave well resolved, characteristic (1→3)-β-d glucan fibre diffraction diagrams with the glucan molecular orientation parallel to the longer axis of the granule. Hydrated electron diffraction patterns with better resolution were obtained from thinner granules by examination at low temperatures of quench-frozen specimens. In this case, the pattern indexed on an hexagonal system with a = b= 1·55 + 0·01 nm and c (fibre axis) = 1·86 ± 0·01 nm. Sections of embedded granules provided single crystal-like electron diffraction patterns corresponding to various sections of the reciprocal lattice of (1→3)-β-d glucan (including the hko section). Finally, by scanning electron microscopy, it was shown that the granules swell on contact with water and take up a characteristic ribbed, pumpkin-like shape.     

An experimental method for calibration of the plasmon mean free path

Transmission electron microscopy specimens in the form of elongated, conical needles were made using a dual‐beam focused ion beam system, allowing the specimen thickness to be geometrically determined for a range of thickness values. From the same samples electron energy loss maps were acquired and the plasmon mean free path (λ) for inelastic scattering was determined experimentally from the measured values of specimen thickness. To test the method λ was determined for Ni (174 ± 17 nm), α‐Al₂O₃ (143 ± 14 nm), Si (199 ± 20 nm) and amorphous SiO₂ (238 ± 12 nm), and compared both to experimental values of λ taken from the literature and to calculated values. The calculated values of λ significantly underestimate the true sample thickness for high accelerating voltages (300 kV) and large collection angles. A linear dependence of λ on thickness was confirmed for t/λ < 0.5–0.6, but this method also provides an approach for calibrating λ at sample thicknesses for which multiple scattering occurs, thus expanding the thickness range over which electron energy loss spectroscopy can be used to determine the absolute sample thickness (t/λ > 0.6). The experimental method proposed in this contribution offers a means to calibrate λ for any type of material or phase that can be milled using a focused ion beam system.     

Using fractal dimensions to characterize atomic force microscope images of epoxy resin fracture surfaces

Atomic force microscopy was used to obtain images of the fracture surface of a tri-ethylene-tetramine and 4,4 (methyl thylidene) epoxy resin. Images were obtained in the mirror, mist, and hackle regions of each sample. Fractal dimensions were calculated from the images using the box dimension and contour analysis method. The box dimension fractal dimension increment for all regions on the fracture surfaces were determined to average 0.26 ± 0.06 and the contour analysis fractal dimension increment were determined to average 0.46 ± 0.05. The box dimension technique is shown to provide the “true” fractal dimension of the surface. The fractal dimension measurements for all three regions indicated that the fracture surface was self-affine and possibly self-similar.     

Reflectance confocal‐laser‐scanning microscopy in vivo assessments of cigarette‐induced dynamic alterations of cutaneous microcirculation on histomorphological level

Objective: Until now, high resolution reflectance confocal‐laser‐scanning microscopy (CLSM) was used for observation of cutaneous morphology in vivo and in real time. We hypothesized that CLSM also allows observation of dynamic processes of cutaneous microcirculation. Methods: Reflectance CLSM (Vivascope1500; Lucid, Rochester, NY) was performed in 24 young male habitual smokers (23 years, range: 19–26, body mass index 23.9 ± 4.04) with relatively limited cigarette exposure (mean: 3.1 ± 2.4 pack‐years). Eight matched nonsmokers served as controls. The quantitative blood cell flow and the diameter of capillary loops were determined prior (baseline), during, as well as 5 and 10 min after smoking. Results: Baseline value for blood cell flow was 55.50 ± 2.33 cells/min, and decreased over 45% during smoking (30.43 ± 3.76/min; P = 0.02). They were still 22% lower (43.33 ± 2.45/min; P = 0.01) 5 min after smoking and exceeded baseline values 10 min after smoking by 13% (63.00 ± 3.10/min; P > 0.05). The baseline values for capillary loop diameter (9.03 ± 0.22 μm) decreased by 21% (7.18 ± 0.28 μm; P = 0.03) during smoking, remained about 9% (8.23 ± 0.18 μm; P = 0.01) lower 5 min after smoking and exceeded baseline values insignificantly by 4% (9.38 ± 0.28 μm; P > 0.05) 10 min after smoking. There were no significant differences to the controls. Conclusion: Reflectance CLSM enables qualitative and quantitative observation of dynamic processes of cutaneous microcirculation on histomorphological level. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

室温辐射探测器用CdZnTe晶体生长及其器件制备

采用改进的垂直布里奇曼法生长了直径为60 mm的碲锌镉晶体,晶体利用率达到70%以上。晶体中Te沉淀/夹杂密度小于1×10-3cm2,电阻率达到4×1010Ω.cm。利用得到的晶体制备了平面型单元探测器,测量了对不同能量射线的分辨率,其中对241Amγ能谱的分辨率达到4.7%,对137Cs能谱的分辨率为4.2%。采用Hecht公式对探测器收集效率与偏压的关系进行了拟合,得到电子的迁移率与寿命乘积值达到2.3×10-3cm2/V。     

An improved method for combined autoradiography and histochemistry: the simultaneous detection of 6-3H thymidine and acid phosphatase activity in cryostat sections of mouse thymus and duodenum

A new method is described that enables the simultaneous detection of 6-³H thymidine incorporation and acid phosphatase activity in the same tissue section. Histochemically, naphthol AS B1 released by tissue based acid phosphatase activity from the substrate naphthyl AS B1 phosphoric acid is coupled with a range of diazonium salts to produce insoluble azo dyes. The azo dye tests result in a particulate localization of lysosomal acid phosphatase and also label diffuse sources associated with cell death. The tests selected permit the application of photographic emulsion without the necessity of an inert barrier layer to separate the emulsion from the histochemically treated cryosections. The localization of 6-³H thymidine incorporation and cell death in mouse thymus and duodenum is demonstrated and comparative counts estimating the distribution of 6-³H thymidine incorporation and hydrolase labelled cell death in the thymus are presented. Young mouse thymus (5 weeks) was found to contain 1·36 ± 0·12% dying cells and 6·78 ± 0·03% thymidine incorporating cells, whilst old mouse thymus (53 weeks) was found to contain 2·34 ± 0·6% dying cells and 5·29 ± 0·37% thymidine incorporating cells.     

The determination of the 1/2<lll> {110} antiphase boundary energy of NiAl

A value for the 1/2 <111> {110} antiphase boundary (APB) energy in stoichiometric NiAl has been obtained by employing the weak-beam technique to resolve the separation of the two 1/2 <111> partial dislocations composing the <111 superdislocation. A separation of 4·5 nm is obtained corresponding to an APB energy of 200 ± 40 mJ m^?2 (erg cm²).     

Surface structures of YBa2Cu3O7-x,BiCa1·7Sr0·7Cu2Ox and TlCaBaCuO4·5±x investigated by scanning tunnelling microscopy

Surface structures of the high-T_c superconductors YBa₂Cu₃O_7-x, BiCa_1·7Sr_0·7Cu₂O_x and TlCaBaCuO_4·5±x have been investigated with scanning tunnelling microscopy. The observed features include well-organized stripe corrugations in Y-Ba-Cu-O as well as orientated flake-like structures and steps with various heights in the Bi- and Tl-compounds. These observations suggest strong local variations of the elastic and electronic properties of the investigated materials.