A temperature controlled chamber to allow observation and measurement of uptake of fluorochromes into live cells

A temperature controlled cell chamber is described which permits phase-contrast and fluorescence microscopy of living cells. It is shown how the cellular uptake of fluorochromes can be investigated both photographically and photometrically. Some sample results are presented for the uptake of Evans Blue.     

Sinusoidal endothelial cells of the liver: Fine structure and function in relation to age

Liver endothelial cells form a continuous lining of the liver capillaries, or sinusoids, separating parenchymal cells and fat-storing cells from sinusoidal blood. Liver sinusoidal endothelial cells differ in fine structure from endothelial cells lining larger blood vessels and from other capillary endothelia in that they lack a distinct basement membrane and also contain open pores, or fenestrae, in the thin cytoplasmic projections which constitute the sinusoidal wall. This distinctive morphology supports the protective role played by liver endothelium, the cells forming a general barrier against pathogenic agents and serving as a selective sieve for substances passing from the blood to parenchymal and fat-storing cells, and vice versa. Sinusoidal endothelial cells, furthermore, significantly participate in the metabolic and clearance functions of the liver. They have been shown to be involved in the endocytosis and metabolism of a wide range of macromolecules, including glycoproteins, lipoproteins, extracellular matrix components, and inert colloids, establishing endothelial cells as a vital link in the complex network of cellular interactions and cooperation in the liver. Fine structural studies in combination with the development of cell isolation and culture techniques from both experimental animal and human liver have greatly contributed to the elucidation of these endothelial cell functions. Morphological and biochemical investigations have both revealed little changes with age except for an accumulation of iron ferritin and a decrease in the activities of glucose-6-phosphatase, Mg-ATPase, and in glucagon-stimulated adenylcyclase. Future studies are likely to disclose more fully the role of sinusoidal endothelial cells in the regulation of liver hemodynamics, in liver metabolism and blood clearance, in the maintenance of hepatic structure, in the pathogenesis of various liver diseases, and in the aging process in the liver.     

Fine structure and function of kupffer cells

Kupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood. The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused-fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm-like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle-coated micropinocytosis for fluid-phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self-replication.     

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

Six-color segmentation of multicolor images in the infection studies of Listeria monocytogenes

Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis.     

Vacuolar sequestration of fluorescent probes in plant cells: a review

The use of fluorescent probes as indicator and tracer molecules is becoming an important aspect of plant cell biology. In many cases the dye, whether introduced directly into the cytosol or sequestered by the cell from its external environment, is preferentially transferred to the vacuole. In the light of increasing evidence for endocytosis in plant cells, the sequestration of high-molecular-weight fluorescent dextrans and the membrane-impermeant dye Lucifer Yellow-CH into the vacuole has been cited as evidence supporting the presence of a fluid-phase endocytic pathway. In this review we consider these recent reports of vacuolar sequestration in the light of new evidence arising on the mechanisms underlying dye uptake.     

The use of a simple method to avoid cell shrinkage during SEM preparation

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO₄-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO₄, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.     

SPC技术在小批量多品种短期生产中的应用

本文分析了传统SPC技术在小批量多品种短期生产中质量控制所存在的问题,应用数理统计方法．得出一种基于正态过程．采取以数据标准化为基础的Z-MR图．可有效用于小批量多品种下对短期生产实施过程质量控制。     

Increasing the signal-to-noise ratio during joint use of the spectrum extrapolation and splitting methods

The possibility of increasing the signal-to-noise ratio by the method of echo-signal spectrum splitting jointly with spectrum extrapolation is considered. The essence of the method proposed is that the echo-signal spectrum known within a given frequency range (f _min, f _max) is divided into several subranges (f _min ⁱ , f _max ⁱ ). The construction of the AR model of the echo-signal spectrum allows the spectrum extrapolation from each subrange by an interval (f _min ^e , f _max ^e that far exceeds the initial interval. This means that, with the use of one echo signal, a set of signals can be obtained, the adding together of which increases the signal-to-noise ratio and ensures an ultrafine beam resolution for flaw images. The presented results of processing echo signals, which were obtained in numerical and model experiments, confirm the efficiency of the proposed technique for processing echo signals.     

Analysis of changes in optical fibers during arc-fusion splicing by use of quantitative phase imaging

A non-interferometric imaging technique in conjunction with Abel inversion is used to directly and quantitatively examine the changes in optical fibers due to the heating produced during arc-fusion splicing as a function of fusion arc parameters. Phase images in the vicinity of a fusion splice are obtained using Quantitative Phase Microscopy, allowing the refractive-index change to be reconstructed with high spatial resolution. This simple, nondestructive method confirms that, for a fixed arc current, while the fusion time increases, the refractive-index of both fiber cores within the fusion region decreases in magnitude, the core region broadens, and the axial gradient decreases.     

用原子力显微镜研究阿莫西林对沙门氏菌和单核增生性李斯特菌的抗菌活性

目的:探测阿莫西林作用于沙门氏菌(G)和单核增生性李斯特菌(G~+)后2种菌体的形貌和生物力学特性的变化,探讨阿莫西林的抗菌活性和抗菌机理。方法:通过平板菌落计数法测细菌的失活率,利用原子力显微镜(AFM)对药物作用后细菌的表面形貌及细胞的硬度、粘附力做定性和定量分析。结果:平板菌落计数得,25μg/mL的阿莫西林作用1h后,沙门氏菌的失活率较李斯特菌的失活率大。AFM测量显示,与低浓度阿莫西林作用后,沙门细菌表面出现孔洞,而李斯特菌表面出现裂缝,力曲线测量显示,药物作用后针尖和细胞壁之间的粘附力明显增加,而杨氏模量(E)显著降低。结论:结合AFM图像可知形貌与生物力学特性的变化反映细胞壁的变化,细胞壁的成分由均一性变为异质性从而导致细菌的粘附力增加即F_(native)E_(amoxicillin)通过以上分析进一步探讨阿莫西林的杀菌机理和不同的细菌对阿莫西林的敏感程度。这些AFM数据为阿莫西林的临床应用提供可视化的数据支持。     

Studying sources of acoustic emission during the cooling of a weld seam with the use of cluster analysis

Acoustic-emission (AE) signals localized during cooling of a weld seam were analyzed. The hazard degree of flaws (faulty fusions, cracks in the root of a weld seam, etc.) was studied using cluster analysis in the parameters of AE signals. Flaws were simulated via introduction of titanium and duralumin inserts into a weld seam. The results of metallographic investigations of specimens with artificial and actual flaws were analyzed. Localization of AE signals was performed for the specimens studied, and a time distribution taking into account the clustering of the total count and energy of AE signals was constructed.     

Visualization of flagellar interactions on bacterial carpets

Methods for the in-depth study of the physics of microscale actuation of microfluidics environments by flagellated bacteria 'teamsters' have been developed. These methods, which include single and multi-colour fluorescent labelling and electron microscopy allow for the analysis of the effect that individual flagellar filaments have on bacterially driven microstructures, and allow for the investigation of the interaction and coordination of flagellar filaments of neighbouring bacteria on densely packed monolayers of bacteria, 'bacterial carpets'. We show that the flagella of bacteria that are immobilized on a surface often interact with each other, and that the flagella of these bacteria do not often form multi-flagella bundles that are aligned with the cell body.     

Microinjecting FM4-64 validates it as a marker of the endocytic pathway in plants

The amphiphilic dye FM4–64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4–64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4–64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4–64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4–64 interacts with.     

Improved preservation of bacterial exopolymers for scanning electron microscopy

Two methods of sample preparation, originally developed for transmission electron microscopy and better at preserving bacterial exopolymers than the traditional fixation with glutaraldehyde, were adapted for scanning electron microscopy. The first involves a glutaraldehyde-lysine mixture as a fixative. The second, based on the use of polycationic ferritin, was modified to include a post-fixation step with either glutaraldehyde or a glutaraldehyde-lysine mixture. These techniques were found to improve considerably the preservation of exopolymers secreted by three bacterial strains.     

Atomic force microscopy of a hydrated bacterial surface protein

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.     

Rapid estimation of bacterial antibiotic susceptibility with flow cytometry

Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15°). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2–5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.