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.     

The use of adhesive-coated grids for the X-ray microanalysis of dry-cut sections in the TEM

Sections cut dry for the X-ray microanalysis of diffusible elements were fixed to adhesive-coated single fine-bar grids. The drawbacks of folding grids normally used for this purpose can thus largely be avoided.     

Elemental concentrations in air-exposed and vacuum-stored cryosections of rat liver cells

Elemental concentrations in different compartments of cryosections of isolated rat liver cells cryotransferred and freeze-dried were compared with those obtained after storage under vacuum for 12 or 60 h and after exposure to room air for 2 min. Poorer image contrast and segregation artefacts are frequently found in air-exposed sections, together with a slight but significant decrease of the K concentration in the cytoplasm and an increase of the S concentration in the liver cell nuclei and the extracellular medium. Extreme distortions of both ultrastructure and elemental distributions are observed if the sections are even slightly colder than the surrounding atmosphere. While storage of frozen-dried cryosections under vacuum for less than 12 h does not lead to alterations in the sections, gross changes are found both in morphology and elemental distribution in sections stored under vacuum for about 60 h. Long-time vacuum storage of frozen-dried cryosections is, therefore, not recommended.     

Suggestions for the quantitative X-ray microanalysis of thin sections of frozen-dried and embedded biological tissues

When a microregion in a thin section of frozen-dried and embedded tissue is analysed by the conventional electron-probe X-ray continuum-normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a ‘tag’ element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measurable, and the X-ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze-drying or by incomplete replacement of water by embedding medium. The same X-ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry-mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.     

Optical disector counting in cryosections and vibratome sections underestimates particle numbers: effects of tissue quality

Optical disector counting is currently applied most often to cryosections, followed in frequency by resin-embedded tissues, paraffin, and vibratome sections. The preservation quality of these embedding options differs considerably; yet, the effect of tissue morphology on numerical estimates is unknown. We tested whether different embedding media significantly influence numerical estimates in optical disector counting, using the previously calibrated trochlear motor nucleus of hatchling chickens. Animals were perfusion-fixed with paraformaldehyde (PFA) only or in addition with glutaraldehyde (GA), or by Methacarn immersion fixation. Brains were prepared for paraffin, cryo-, vibratome- or celloidin sectioning. Complete penetration of the thionin stain was verified by z-axis analysis. Neuronal nuclei were counted using an unbiased counting rule, numbers were averaged for each group and compared by ANOVA. In paraffin sections, 906 +/- 12 (SEM) neurons were counted, similar to previous calibrated data series, and results obtained from fixation with Methacarn or PFA were statistically indistinguishable. In celloidin sections, 912 +/- 28 neurons were counted-not statistically different from paraffin. In cryosections, 812 +/- 12 neurons were counted (underestimate of 10.4%) when fixed with PFA only, but 867 +/- 17 neurons were counted when fixed with PFA and GA. Vibratome sections had the most serious aberration with 729 +/- 31 neurons-a deficit of 20%. Thus, our analysis shows that PFA-fixed cryosections and vibratome sections result in a substantial numerical deficit. The addition of GA to the PFA fixative significantly improved counts in cryosections. These results may explain, in part, the significant numerical differences reported from different labs and should help investigators select optimal conditions for quantitative morphological studies.     

A simple microtome capable of cutting sections of plastic-embedded material down to 1 μm in thickness

Details of the design of an easily constructed microtome are presented. The instrument uses glass knives to cut sections down to about 1 μm in thickness, and is suitable for the examination of plastic-embedded material.     

Observations on the production of frozen-dried thin sections for electron microscopy using unfixed fresh liver, fast-frozen without cryoprotectants.

Thin sections of unfixed liver, fast-frozen without cryoprotectants, have been cut using conditions under which momentary thawing of the sections is unlikely to occur. A transfer stage which facilitates this procedure is described. Sections show hole damage probably due to ice-crystal formation during the freezing process and have well defined edges, but despite hole damage, some morphological features of the cell are discernible. Presumptive mitochondria appear smaller in frozen sections than in conventional Araldite sections. Sections devoid of hole damage have indistinct edges and are presumed to have undergone transient thawing. Carbon coating of freeze-dried sections to exclude atmospheric moisture during transference of sections to the electron microscope (EM) appear unnecessary as regards preservation of morphological structure. The results are discussed in relation to the limitations of the method and the potential value of the technique.     

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.     

K-shell ionization by protons: A quantitative comparison between published cross sections and theories

The cross sections for K-shell ionization by protons available in the literature are compared to each other and to the ECPSSR theory by Brandt and Lapicki. For low scaled velocities ξ, we find that a “Coulomb correction factor” C_K = exp(−λx) can explain the data, with λ increasing with target atomic number Z₂, where the variable x is proportional to ξ⁻³. This factor C_K probably contains a residual part of the relativistic correction.For medium to high scaled velocities ξ, we average normalized experimental ionization cross sections s = σ_exσ_ECPSSR over all targets, within various small intervals Δξ. The average cross sections are found to agree well with σ_ECPSSR. After rejecting the data sets containing the most discrepant data, using a statistical criterion, the remaining data are almost compatible with each other. No clear evidence of a dependence of s upon Z₂ found. Empirical reference cross sections for Cu, Ag and Au targets are calculated at a few selected energies.When comparing four different SCA calculations for the ionization probability vs. impact parameter, for 2 MeV protons on Cu, we find that the curves disagree by at most 30% and that they bracket the two sets of data available.     

Y形和Y环形单元特性的实验对比研究

采用镀膜和光刻技术设计并制备出了2种衬底厚度不同的Y孔单元和Y环孔单元的FSS,在0°和45°入射角下分不同的极化波进行了测试,经对比研究发现,在大角度入射下Y环单元FSS比Y孔单元FSS有更为稳定的中心频率,更窄的带宽;Y环单元FSS对垂直和水平极化波的适应性更强,滤波特性较Y孔单元FSS更明显;介质厚度的变化对Y孔中心频率、带宽的影响要比Y环单元FSS的大得多。所以,从中心频率、带宽随介质衬底厚度、入射角度、不同极化方式的变化情况看,Y环单元比Y孔有更稳定的性能。     

A section press and low elemental support for enhanced preparation of freeze-dried cryosections

A press- and specimen holder system is described, whereby thin, freeze-dried cryosections can be obtained more conveniently and with greater protection against contamination and harmful temperature fluctuations. The use of a grid and retainer assembly as the basic working unit, greatly facilitates low temperature work particularly when delicate grids are preferable from an analytical point of view. A new press has been constructed in order to keep the sections permanently pressed during freeze-drying and rewarming. The press also protects the sections during transfer to an external freeze-dryer.     

A finite element method comparison of wear in two metal-on-metal total hip prostheses

The contact mechanics of two metal-on-metal (MOM) total hip prostheses was studied by means of the finite element method (FEM). The purpose of the work was to compare two total hip replacements (Durom and Metasul) with regard to the amount of wear debris released. Wear on the bearing surfaces was evaluated following Reye hypotheses from the pressure distribution, computed by means of three-dimensional FEM models; an approximate analytical model based on Hertz contact theory has also been developed and discussed. The results show that in the dry friction condition the Durom joint releases almost twice as much wear volume as produced by the Metasul joint. Therefore, while Durom implants can improve hip stability by increasing the prosthetic impingement-free range of motion (PIF-ROM), Metasul prostheses can be a valuable solution whenever wear represents a critical choice factor.     

一种金属元素盲样测量结果修正方法

以Na、Cu和Li为例,介绍了一种金属元素盲样测量结果修正方法,并以实验室间比对的形式进行了验证。实验选择稳定性高的方法,用基体匹配法消除干扰,用相近浓度标准物质的测量结果修正盲样测量值,并计算扩展不确定度。对于Na、Cu和Li浓度参考值（K=2）分别为0.86±0.02 mg/L、95.8±1.9μg/L、1.35±0.03mg/L的盲样,分析结果（K=2）分别为855.5±6.5μg/L、97.4±0.6μg/L、1.344±0.007 mg/L。实验室间比对显示：测量结果均为非粗大误差可疑数据,En值评价结果有效,ZR值评价结果满意。方法满足盲样测量需求,可供各实验室参考。     

岸边集装箱起重机门架结构的有限元分析与比较

针对常见的复合型门架结构和单斜撑型门架结构,利用ANSYS软件对岸桥集装箱起重机钢结构进行有限元建模,分析比较其应力、变形和模态振动等方面情况.分析结果显示在各项性能参数相同的情况下,由复合型门架结构改变为单斜撑型门架结构,其应力、应变和固有特性的变化不是很大;提升联系横梁,采用单斜撑型门架结构可行,它可以节约钢材、降低成本、提高横梁下运行净空高度.     

Diffusible ion localization by ion microscopy: a comparison of chemically prepared and fast-frozen,freeze-dried,unfixed liver sections

The necessity of low temperature preparative procedures for diffusible ion localization using the ion microscope has been established with rat liver as the test material. Using conventional fixation procedures, significant ion loss and redistribution were observed which exceeded the 1 μm lateral resolution of the direct imaging ion microanalyser. The compositional morphology evidenced in chemically prepared versus fast-frozen, freeze-dried, unfixed material was compared and evaluated in terms of ion retention, ion redistribution and morphological integrity. A mass spectral analysis of both preparations has been presented and discussed with regard to spectral interferences and elemental sensitivities. Ion images of sodium and potassium were recorded for chemically prepared liver sections and compared with the ion distributions obtained for unfixed, fast-frozen, freeze-dried 1 μm thick sections. The magnitude of ion loss during preparation of samples removed at different stages in the course of chemical processing was determined by atomic absorption and flame emission spectrophotometry.     

Effect of specimen preparation and section transfer techniques on the preservation of ultrastructure,lipids and elements in cryosections

Cryofixation, cryoultramicrotomy, and proper transfer of the cryosections into the electron microscope are important for the preservation of good ultrastructure and the measurement of subcellular elemental distributions. These techniques are applicable to tissue systems which can be rapidly frozen so that minimal to no ice damage occurs during the cryofixation step. For the transfer step we have compared the cryotransfer of hydrated sections and subsequent freeze-drying in the electron microscope with the transfer of sections into an external freeze-dryer, followed by exposure to room temperature and humidity before introduction into the electron microscope. The use of a cryotransfer stage for section transfer from the cryoultramicrotome to the electron microscope and low temperature observation of the thin sections avoids the potential problem of rehydration damage to freeze-dried sections as well as provides protection from the possibility of melting of the lipids in the sections. Both of these problems may lead to loss of in situ elemental distribution and morphology. In this report, observations are presented which show the damaging effects of temperatures above 273 K on ultrastructure due to lipid melting in tissues with high lipid content and the redistribution of elements which can be encountered when thin sections become inadvertantly rehydrated.