The intracellular distribution of ions and water in rat liver and heart muscle

Local dry mass concentrations of intracellular compartments in rat heart muscle and liver cells were estimated by quantitative electron microscopy and X-ray microanalysis of ultrathin frozen-dried cryosections. The results were used to calculate elemental concentrations per litre of compartment water from the X-ray microanalytical data. Water fractions were between 80.3 ± 1.3% of wet weight in the decondensed chromatin and only 45.1 ± 1.7% in mitochondria of liver cells. The lowest water fraction in heart muscle cells was also found in mitochondria. The ionic concentrations found in the cytoplasm of liver cells and in the myofibrils are in accord with the electroneutrality rule and in osmotic equilibrium with the extracellular concentrations. The concentrations of Na, K, Cl and P both in the cytoplasm and in the regions of decondensed chromatin within the nuclei were found to be equal. However, in regions of condensed chromatin K⁺ concentrations were found to be much higher than expected for a Donnan distribution of ions free in solution. Most probably the activity coefficient for K⁺ is lower in the condensed chromatin than in the decondensed or in the cytoplasm. The same holds true for the A-band as compared to the I-band in heart muscle cells. A sequestration of K⁺ was measured also in the rough endoplasmic reticulum (RER) of hepatocytes. The Cl^? concentration in mitochondria both in heart muscle and liver cells has been measured far in excess of what might be expected from a Nernstian distribution. A coupled inward Cl^? transport in mitochondria must, therefore, be assumed.     

Electron probe X-ray microanalysis of intracellular element concentrations in cryosections in the presence of changes in cell volume

The interpretation of element concentration data for X-ray microanalyses of biological tissues, which are subjected to some experimental treatment, can be complicated by changes in cell volume and total cell dry matter induced by the treatment. We have examined the manner in which such changes would affect the values measured in frozen-dried cryosections of soft tissues, and how they may be taken into account in the interpretation of the results. The element content (mass per unit dry weight) measured by the peak-to-continuum or Hall method is independent of changes in cell volume, but is sensitive to a change in the local dry mass. Conversely, intracellular concentrations in terms of mass per unit volume, as determined by the peripheral or internal standard technique, are dependent on volume changes but independent of dry mass. The estimated dry weight fraction is affected by changes in both volume and dry mass. The results obtained from both quantification methods can therefore provide information on the combination of changes in cellular element levels, volume and total dry mass that may occur following the experimental treatment. In a study of the late effect of the drug cisplatin on electrolyte concentrations in kidney proximal tubules, both quantification methods have been used to obtain wet weight and dry weight concentrations. By applying the above considerations, the analytical results have been interpreted as a combination of changes in element levels and a shrinkage of the tubule cells. Cell shrinkage was confirmed by morphometric analysis of tubular cross-sections.     

Vitrification of articular cartilage by high-pressure freezing

For more than 20 years, high-pressure freezing has been used to cryofix bulk biological specimens and reports are available in which the potential and limits of this method have been evaluated mostly based on morphological criteria. By evaluating the presence or absence of segregation patterns, it was postulated that biological samples of up to 600 μm in thickness could be vitrified by high-pressure freezing. The cooling rates necessary to achieve this result under high-pressure conditions were estimated to be of the order of several hundred degrees kelvin per second. Recent results suggest that the thickness of biological samples which can be vitrified may be much less than previously believed. It was the aim of this study to explore the potential and limits of high-pressure freezing using theoretical and experimental methods. A new high-pressure freezing apparatus (Lei?a EM HPF), which can generate higher cooling rates at the sample surface than previously possible, was used. Using bovine articular cartilage as a model tissue system, we were able to vitrify 150-μm-thick tissue samples. Vitrification was proven by subjecting frozen-hydrated cryosections to electron diffraction analysis and was found to be dependent on the proteoglycan concentration and water content of the cartilage. Only the lower radical zone (with a high proteoglycan concentration and a low water content compared to the other zones) could be fully vitrified. Our theoretical calculations indicated that applied surface cooling rates in excess of 5000 K/s can be propagated into specimen centres only if samples are relatively thin (<200 μm). These calculations, taken together with our zone-dependent attainment of vitrification in 150-μm-thick cartilage samples, suggest that the critical cooling rates necessary to achieve vitrification of biological samples under high-pressure freezing conditions are significantly higher (1000–100 000 K/s) than previously proposed, but are reduced by about a factor of 100 when compared to cooling rates necessary to vitrify biological samples at ambient pressure.     

AG2.0型超声波蒸发传感器遇低温结冰时记录的处理

文章分析了3a马尔康国家基准气候站AG2.0型超声波蒸发传感器观测数据,结果表明:蒸发传感器遇低温结冰时段自动观测数据偏大。对1d中部分时间段内蒸发水结冰、融化以及冬季全天结冰期很短的观测数据进行了分析,分析认为数据偏大主要是因为蒸发桶和测量筒的材料物理性质以及测量的水体面积不同。最后提出了数据记录处理建议。     

水中氦同位素的质谱分析

本文是关于水中氦浓度及其同位素质谱分析技术的研究报告，使用特制的玻璃瓶采集水样，真空脱气率大于９８％，经净化析出Ｈｅ和Ｎｅ送质谱计测量。以空气氦为标样，采用“ＡＢＡ”峰高比分析技术，对蒸馏水，泉水，西太平洋浅层海水等样品的氦浓度同位素组成进行了测试，该项技术已在探讨西太平洋浅层海水氦同位素分布的研究中获得了成功的应用。     

C波段制冷接收机充气系统

充气系统是给制冷接收机干空气腔体提供新鲜干燥空气、排出陈旧气体以防止水汽在真空窗表面遇冷凝结而影响观测的气动系统。某26 m射电望远镜C波段制冷接收机的馈源在杜瓦外部,故该接收机的充气系统将馈源设计为干空气腔体,通过充气机提供干燥空气,使用气体缓冲器作为辅助气源用于在充气机停止工作时补充提供干燥空气,以小的泄漏排出馈源腔体内的陈旧气体。该气动系统能很好地避免C 波段制冷接收机真空窗口表面结冰,在自动控制下的充气系统也可以很好地补偿腔体内压的变化,使得整个充、排气过程更加稳定,真空窗口表面保持干燥。     

Cylindrical diameters method for calibrating section thickness in serial electron microscopy

An estimate of section thickness is required for measuring structures in serial section microscopy. Mean section thickness is estimated reliably by averaging the ratios of the diameters of cylindrical objects, such as mitochondria, to the number of sections they span. This cylindrical diameters method improves the accuracy of section thickness as inferred from the colour of sections floating in water. The cylindrical diameters method gives the same answer as that obtained by the minimal folds method. It is preferable because it can be done in a series that has no folds that can distort and obscure the objects that are being measured.     

Application of ISO4359 for discharge calculation in a narrow flume

Flumes with either width contractions or raised beds to force modular flow conditions (i.e. a transition from sub- to super-critical flow) are a widely used instrument for the measurement of flow rate. The conservation of mass, written as the continuity equation, and the conservation of energy are combined with the existence of a critical depth point to derive a theoretical equation for the discharge as a function of the upstream water depth. This derivation requires a number of assumptions regarding the flow both upstream and in the throat of the contraction. The international standard covering the use of such flumes, ISO4359, places restrictions on the upstream position at which the water depth should be measured, which this work shows to be unnecessary, at least for the small (100 mm throat width) flumes examined. The assumption that critical depth occurs at the end of the flume throat is also shown to be incorrect, but has negligible effect on discharge calculation using the ISO4359 method.     

Electron cryo-microscopy of vitrified bulk biological specimens: ideal and real structures of water—lipid phases

Lipid-water mixtures were studied by X-ray cryodiffraction in order to assess the structural changes during freezing. We show that the water of aqueous lipid phases, in the concentration range of 10–30% (water weight/total weight), is vitrified by high-pressure freezing. Vitrified lipid phases can be cryo-sectioned and imaged by electron cryomicroscopy. Both the ideal or average and the real or local structures of the lipid mixtures can be studied at a resolution better than 2 nm. While the average structure of the lipid phases is in good agreement with that determined by X-ray diffraction, the local structure reveals features that might play an important role in the function of biological membranes such as in endo- and exocytosis.     

Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy

A method is described for the cryofixation of biological specimens for ultrastructural analysis and immunocytochemical detection studies. The method employs plunge freezing of specimens in a sealed capillary tube into a cryogen such as liquid propane or liquid nitrogen. Using this method a number of single-cell test specimens were well preserved. Also multicellular organisms, such as Caenorhabditis elegans , could be frozen adequately in low ionic strength media or even in water. The preservation of these unprotected specimens is comparable to that achieved with high-pressure freezing in the presence of cryoprotectant. The results are explained by the fact that cooling of water in a confined space below the melting point gives rise to pressure build-up, which may originate from the conversion of a fraction of the water content into low-density hexagonal ice and/or expansion of water during supercooling. Calculations indicate the pressure may be similar in magnitude to that applied in high-pressure freezing. Because the specimens are plunge cooled, suitable cryogens are not limited to liquid nitrogen. It is shown that a range of cryogens and cryogen temperatures can be used successfully. Because the pressure is generated inside the specimen holders as a result of the cooling rather than applied from an external source as in high-pressure freezing, the technique has been referred to as self-pressurized rapid freezing.     

Thin frozen-dried cryosections and biological X-ray microanalysis

The application of X-ray microanalysis to problems of cell physiology required the development of methods to retain diffusible substances within the subcellular compartments that they occupied in vivo. Several groups have developed methods of rapidly freezing small samples in ways that minimize mechanical traumae and ice crystal formation. This provides a narrow zone from which cryosections, believed to be representative of the in vivo distribution of electrolytes, can be cut. The production of thin (less than 0·5 μm) cryosections that are apparently free of diffusion can be routinely performed when temperature parameters are kept below 173 K. Efficient cryosectioning requires several modifications to commercially available machines, in order to improve the ease and reliability with which various manipulations can be carried out. Initial attempts to localize calcium at the subcellular level were disturbed by the use of mechanically damaged specimens and by insufficiently cold conditions in the cryochamber. Such sections indicated that mitochondria were calcium-rich organelles. When tissue freezing and cryosectioning were performed under optimized conditions, mitochondrial calcium was so low as to be quantifiable only with difficulty. Available microanalytical results show that ER-rich cytoplasm and terminal cisternae of the sarcoplasmic reticulum seem to contain higher levels of calcium than mitochondria. Nuclei and secretory granules also contain more calcium than mitochondria.     

Freezing of aqueous specimens: an X-ray diffraction study

The effects on water of two cooling methods, immersion in a liquid cryogen and high-pressure freezing, were studied by X-ray cryodiffraction on different sucrose solutions. The nature of the ice formed by each method depends on both the sucrose concentration and the specimen thickness. In order to compare the two methods, we mainly studied specimens having a thickness of 0.2 mm. Under these conditions, freezing by immersion gives rise to hexagonal (IH), cubic (IC) and amorphous (IV) ices when the sucrose concentration (weight/weight) has a value within the range 0–30%, 30–60%, 60% and higher, respectively. The temperature of the phase transitions IV–IC, IC–IH depends on the sucrose concentration. High-pressure freezing gives rise to two specific forms of ice: an amorphous and a crystalline ice (ice III). Ice III is observed when pure water samples are high-pressure frozen provided that the sample temperature does not rise above −150 °C. Above this temperature, ice III transforms into hexagonal ice. Amorphous ice is formed when the sucrose concentration is higher than 20%. The amorphous ice formed under high pressure has a similar, but not identical, X-ray diffraction pattern to that of amorphous ice formed at atmospheric pressure. While the X-ray diffraction pattern of amorphous ice formed at atmospheric pressure (IV) shows a broad ring at a position corresponding to 0.37 nm, that of high-pressure amorphous ice (IVHP) shows a broader ring, located at 0.35 nm. IVHP presents a phase transition (IVHP–IV) at temperatures that depend on the sucrose concentration. We also observed that some precautions have to be taken in order to minimize the alcohol contamination of high-pressure frozen samples. The ice-phase diagram presented in this paper should be taken into account in all methods dedicated to the structural study of frozen biological specimens.     

保持制冷接收机真空窗口干燥的充气系统

微波接收机系统是射电天文观测不可或缺的信号接收设备。通过制冷可以很大程度上降低接收机的噪声温度,从而提高接收机的灵敏度。在接收机制冷后,杜瓦内外温差达200 K以上。由于1.3 cm波段接收机的特殊设计,空气中的水汽会在真空窗处与低温密封薄膜直接接触从而遇冷凝结,这将会影响接收机的接收性能。因此,1.3 cm波段接收机在真空窗外围设计一个干空气腔体,通过充气系统给该腔体时刻注入新鲜、干燥的空气,用小的泄漏来排除腔体内部陈旧气体。这样,真空窗与外界就始终被干燥的空气隔离开,从而避免了水汽在该位置的遇冷凝结。     

Calibration of transmission electron microscopic mass determination using objects with known mass distribution

An improved calibration procedure is described for dry mass determination methods, that are based on microdensitometry of transmission electron micrographs. Polystyrene latex spheres, that are supposed to have a homogeneous mass distribution, are used for the calibration procedure. By computer fitting of the theoretically expected mass distribution to the distribution measured in a line through the centre of a sphere, a mass calibration factor is obtained. The number of measurements required for a predetermined calibration accuracy can be decreased considerably as compared to other calibration methods. Accurate calibration and reproducible adjustment of the electron optical magnification are required.     

The Friction Reducing Properties of Molybdenum Dialkyldithiocarbamate Additives: Part I — Factors Influencing Friction Reduction

Abstract

Calcium sulfonate complex greases have excellent extreme pressure, antiwear, and anticorrosion properties and are widely applied in rolling bearings, particularly in humid environments. In this article, the shear stability of dry and water-contaminated lubricating calcium sulfonate complex greases is described using a novel aging method. Unlike lithium and polyurea greases, no shear softening is observed for the dry greases due to the good mechanical stability of the particle-like thickener structure. For water-contaminated greases, no water separation was found during the prolonged aging. Instead, a homogeneous water–calcium sulfonate thickener micellar structure is generated. These micelles function as apparent thickeners and effectively increase the thickener concentration, which thickens the grease. This may explain why calcium sulfonate complex grease has excellent water absorption properties.     

Algorithm for calculating the eutectic point with active devices for detecting the formation of ice

Glaze sensors are intended for diagnosing the state of the surfaces of roads and runways of airports and allow determination of the freezing temperature and the concentration of aqueous solutions in the form of film precipitations deposited on the sensor surface. When antifreeze reagents fall on the snow or ice surface, their particles dissolve with the formation of a brine of varying concentration, which has a freezing temperature below that of water. In this case, a glaze sensor solves a double problem-to forecast the freezing temperature of a mixture and register the pavement pickling. The presented algorithm for calculating the eutectic point and concentration of salts allows evaluation of the above pavement characteristics from the sensor readings. Original Russian Text ? K.F. Ivanova, 2009, published in Pribory i Tekhnika Eksperimenta, 2009, No. 3, pp. 123–128.     

A physical principle for development of a glaze sensor as a monitor of the state of the ecosystem

Glaze sensors are designed for diagnostics of glaze-frost states and make it possible to measure the freezing temperature and concentration of solutions in the form of film deposits coating a sensor surface. The physical basis of the operational principles of glaze sensors allows them to be considered as an efficient analyzer of the state of the environment and to be included in the information system of environmental protection that operates as an alarm system of the glaze situation and as a monitor of soil and water area salinization.     

干冰升华式天然气水合物孔底冷冻取样器的研制

针对天然气水合物的赋存特点,研制天然气水合物孔底冷冻取样器。取样器是以干冰为冷冻剂,酒精为助冷催化剂和载冷剂的冷冻方式来实现孔底冷冻岩样。介绍了取样器的技术参数、冷冻方式原理、工作原理和结构特点。     

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-μm thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.     

Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions

The freezing of biological cell suspensions can be understood in terms of ice formation in the external suspension medium and the cellular reactions to the changing environment. Cryomicroscopy allows a quantitative analysis of both categories of phenomena. Besides freezing stages of appropriate thermal design, the components used for that purpose include a microcomputer (PSI 80) based control system, an image analysis system (Intellect 100) and a spectrophotometer (MPV compact). The investigation of extracellular ice formation is focused on the following effects: The redistribution of solutes in the residual liquid and the resulting concentration profiles are determined photometrically or densitometrically. The transitions between various morphologies of the ice–liquid phase boundary (planar–cellular–dendritic) can be related to interface instability theories. With respect to solute segregation, the studies also involve the formation of bubbles from supersaturated gaseous solutes and freezing potentials resulting from the differential incorporation of cations and anions into the solid phase. The interaction between particles or cells and the advancing ice front is determined from critical interface velocities marking the transition between repulsion and entrapment. The effects of freezing on biological cells are studied mainly with blood cells, especially lymphocytes. The water efflux due to osmotical gradients across the membrane yields volume shrinkage curves which are recorded and analysed from video images for various cooling rates. Beyond a certain threshold cooling rate, intracellular ice starts to form, and different crystallization morphologies can be detected. The intracellular crystallization temperatures depend on cooling and warming rates as well as on the presence of penetrating cryoadditives. A fluorescence viability is used to determine the percentage of damaged cells immediately after thawing.