Study of potassium deficiency in cardiac muscle: quantitative X-ray microanalysis and cryotechniques

An imbalance of potassium in cardiac muscle causes an alteration of heart function. The distribution and concentration of potassium in rat papillary heart muscle was studied using cryofixation and X-ray microanalysis. Freeze-dried cryosections and sections of freeze-dried, embedded tissue were analysed. Bulk frozen specimens were freeze-dried either in a vacuum or by a new technique using liquid propane as a cryodehydration medium. These two methods of freeze-drying were tested for elemental retention in other specimens, with comparable results. A potassium concentration of 120 mmol/l was measured in normal myocytes of cardiac papillary muscle compared to 80 mmol/l in myocytes of animals stressed by a temperature of 45°C for 1 h. The presumed physiological significance of the findings is discussed.     

Quick sampling and perpendicular cryosectioning of cell monolayers for the X-ray microanalysis of diffusible elements

A quick sampling and preparation method for freezing of cell monolayers is described. The cells are grown on a large Formvar film supported by a frame of polystyrene. A polyvinylpyrrolidone (PVP) solution is applied to one side of the film forming a flat disc when frozen with a pair of pliers precooled in liquid nitrogen. The PVP solution provides the specimen with sufficient strength and may be used as an elemental standard for absolute quantification if salts of known concentrations are added. Manipulation of the cells prior to freezing is thus restricted to a minimum, which eliminates possible harmful treatments like scraping and centrifugation. The procedure is quickly performed, the freezing being completed within 30 s of the cells having been removed from the culture well. The analytical results reveal low and stable Na: K ratios. Our results confirm that cells in vitro are comparable to cells in vivo with respect to elemental composition.     

The effect of aluminium coating on elemental standards in X-ray microanalysis

The standardisation of frozen hydrated bulk biological specimens using gelatin standards is described. The relationship between corrected elemental X-ray counts and ionic concentration was found to be linear, and minimum detectable limits for each element are stated. Variations in uncorrected standard curves were found to be due to changes in aluminium coating thickness. There was an inverse relationship between coating thickness and elemental X-ray counts. The factors causing this are discussed. To avoid errors arising from inconsistent aluminium thickness, experimental material should only be compared with standards of similar aluminium net counts. This can be achieved most easily by mounting and analysing specimen and standard together.     

X-ray microanalysis of cultured keratinocytes: methodological aspects and effects of the irritant sodium lauryl sulphate on elemental composition

Irritant substances have been shown to induce elemental changes in human and animal epidermal cells in situ . However, skin biopsies are a complicated experimental system and artefacts can be introduced by the anaesthesia necessary to take the biopsy. We therefore attempted to set up an experimental system for X-ray microanalysis (XRMA) consisting of cultured human keratinocytes. A number of methodological aspects were studied: different cell types, washing methods and different culture periods for the keratinocytes. It was also investigated whether the keratinocytes responded to exposure to sodium lauryl sulphate (SLS) with changes in their elemental composition. The concentrations of biologically important elements such as Na, Mg, P and K were different in HaCaT cells (a spontaneously immortalized non-tumorigenic cell line derived from adult human keratinocytes) compared to natural human epidermal keratinocytes. The washing procedure and time of culture influenced the intracellular elemental content, and rinsing with distilled water was preferred for further experiments. Changes in the elemental content in the HaCaT cells compatible with a pattern of cell injury followed by repair by cell proliferation were seen after treatment with 3.33 µ m and 33 µ m SLS. We conclude that XRMA is a useful tool for the study of functional changes in cultured keratinocytes, even though the preparation methods have to be strictly controlled. The method can conceivably be used for predicting effects of different chemicals on human skin.     

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.     

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

The effect of some instrument operating conditions on the x-ray microanalysis of particles in the environmental scanning electron microscope

The objective of this investigation was to evaluate the practical effects of electron beam broadening in the environmental scanning electron microscope (ESEM) on particle x-ray microanalysis and to determine some of the optimum operating conditions for this type of analysis. Four sets of experiments were conducted using a Faraday cage and particles of copper, glass, cassiterite, andrutile. The accelerating voltage and chamber pressure varied from 20 to 10 kV and from 665–66 Pa (5.0 to 0.5 torr), respectively. The standard gaseous secondary electron detectors (GSED) and the long environmental secondary dectectors (ESD) for the ESEM were evaluated at different working distances. The effect of these parameters on the presence of artifact peaks was evaluated. The particles were mounted on carbon tape on an aluminum specimen mount and were analyzed individually and as a mixture. Substrate peaks were present in almost all of the spectra. The presence of neighboring particle peaks and the number of counts in these depended upon the operating conditions. In general, few of these peaks were observed with the long ESD detector at 19 mm working distance and at low chamber pressures. More peaks and counts were observed with a deviation from these conditions. The most neighboring peaks and counts were obtained with the GSED detector at 21.5 mm working distance, 10 kV accelerating voltage, and 665 Pa (5.0 torr) chamber pressure. The results of these experiments support the idea that the optimum instrumental operating conditions for EDS analysis in the ESEM occur by minimizing the gas path length and the chamber water vapor pressure, and by maximizing the accelerating voltage. The results suggest that the analyst can expect x-ray counts from the mounting materials. These tests strongly support the recommendation of the manufacturer to use the long ESD detector and a 19 mm working distance for EDS analysis. The results of these experiments indicate that neighboring particles millimeters from the target may contribute x-ray counts to the spectrum.     

Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X-ray microanalysis of cryosectioned and freeze-substituted specimens

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X-ray microanalysis. Directly frozen control and potassium-depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze-substitution in presence of oxalic acid followed by sectioning of resin-embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze-drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of 'dead' nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.     

X-ray microanalysis of ion distribution in frozen salt/dextran droplets after freeze-substitution and embedding in anhydrous conditions

A model system using dextran droplets of different salt solutions either frozen together, or sandwiched together after freezing and then freeze-substituted, embedded and dry sectioned has been investigated by X-ray microanalysis. X-ray maps and spot measur ements taken in transects through the interface of both droplets have shown that the P, K and Ca remain well localized in their original droplet. This validates freeze-substitution as a method for localization of these elements in biological samples. Results with Na were more variable and not always explainable. Success was achieved by the use of super-dry solvents and maintenance of a dry environment at all stages. We emphasize the need to avoid water contamination not only during freeze-substitution but also during sectioning, storage and section transfer to the electron microscope.     

The continuum normalization method for quantification of X-ray spectra in biological microanalysis. 1. Generalized bremsstrahlung production cross-sections and analysis using standards

The thin self-supporting biological specimens used for quantitative X-ray microanalysis are problematical because the sections are most unlikely to be uniform in thickness or density, so the intensities of the characteristic lines alone are not a good measure of composition. The method developed to overcome these problems was introduced by T. A. Hall in 1971 and uses the bremsstrahlung or continuum intensity recorded in the X-ray spectrum to normalize each characteristic line, and hence is frequently referred to as the continuum normalization (CN) procedure.
  Reformulating the CN method of quantification in terms of generalized cross-sections and calculating more accurate values of bremsstrahlung production using a formula allows us a better understanding of the options open to the analyst of biological thin sections by which the errors in the measurement may be reduced. If one chooses to use the original Hall (1971 ) method using Kramers cross-sections, the window measuring the continuum for normalization should be set in the 4–7 keV region for typical scanning electron microscope and microprobe beam energies, 20–40 kV, and above 10 keV for transmission electron microscope energies of 80 kV and above. Although it is clear that peak counts must not contribute to the white count, the window should be as wide as possible to reduce statistical errors.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Surface roughness and the use of a peak to background ratio in the X-ray microanalysis of bio-organic bulk specimens

The use of a net peak intensity and of a peak to background (P/B)-ratio of sulphur and chlorine is examined in the X-ray microanalysis of a 2·4% w/w S bulk standard in Spurr's epoxy resin. In calculating the P/B-ratio, the background intensity is calculated for the same energy region as for the net peak. Analyses were carried out on the flat top of the standard and on the slope running down from the top on the side not facing the X-ray detector. The results obtained for the peak to local background ratios from the top and the slope yielded a relatively small mean deviation (11%) while net peak intensities ultimately were reduced to 7% or less of the initial value for the flat top. This indicated that a peak to local background ratio is to be preferred in the quantitative analysis of bulk specimens which have poorly defined local tilt and takeoff angles. A second advantage is the inherent correction for beam current fluctuations.     

能量色散X射线荧光光谱仪快速测定土壤中Cu、Pb、Zn、Rb、Nb和Th6种元素

直接使用粉末样品,用便携式能量色散X射线荧光光谱仪测定土壤样品中的Cu、、Zn、Pb、Rb、Nb和Th等6种元素。使用土壤、水系沉积物标准样品建立校准曲线,用铑靶Kα线的康普顿散射线作为内标校正基体效应。方法简便、快速、准确。除Th精密度为13%外,各组分分析的精密度(RSD,n=12)均小于6%,完全能满足化探样品测定的要求。     

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: Calibration curves and electron beam sensitivity

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K Kα P/B or Y = (P/B) · Z²/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10–25 kV for Ca standards. K standards were also independent when P/B was corrected for Z²/A. Background counts in the otoconia (B_o) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. B_o was not dependent on the spectra acquisition time, with the exception of B_o under Kα K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z²/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.     

Combined quick freezing,freeze-drying,and embedding tissue at low temperature and in low viscosity resins

The techniques of quick freezing and freeze-drying provide an alternative to the more classical methodologies of chemical fixation and dehydration with organic solvents. It is possible to embed freeze-dried tissue in low viscosity resins, either at room temperature or at subzero temperatures in Spurr's resin or Lowicryl K4M, respectively. The choice of embedding medium affords additional flexibility in postdrying and embedding conditions, since Spurr's resin allows vapor fixation with osmium tetroxide and thermal polymerization. Osmium tetroxide is not recommended for Lowicryl resins, but these media permit polymerization at subzero temperatures with ultraviolet light. Both resins have unique advantages that may be utilized, depending upon the purpose of the embedding. In this paper, we discuss the details of preparing smooth muscle, from rabbit renal artery, by quick freezing and freeze-drying, as well as methods for the embedding of the freeze-dried tissue in both Spurr's resin and Lowicryl K4M. Although we have previously reported the ultrastructure of smooth muscle embedded in Spurr's low viscosity resin, the combination of freeze-drying and infiltration in Lowicryl K4M represents a new approach that allows the elimination of chemical fixation, dehydration with organic solvents, and heat polymerization of the embedding medium.     

Effects of veratrine on skeletal muscle mitochondria: ultrastructural, cytochemical, and morphometrical studies

The alkaloid veratrine is a lipid-soluble neurotoxin, which target voltage-gated Na+ channels for their primary action. Recently, we showed that this alkaloid may cause myonecrosis and evidences suggest mitochondria as one of its cell targets. Herein, we investigate the effects caused by variable concentration of veratrine (250 and 550 microg/mL) on mitochondrial oxygen consumption, respiratory chain enzymes activities, and ultrastructure, combining electron microscopy with cytochemical and biochemical approaches. The results showed different sort of ultrastructural changes, both in isolated and intramuscular mitochondria. Veratrine decreased mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH-d), succinic dehydrogenase (SDH), and cytochrome oxidase (COX) activities, significantly and dose-dependently inhibited the state 3 respiration rate, respiratory control ratio (RCR), and ADP/O on isolated rat skeletal muscle mitochondria, whereas state 4 was unaffected. A tendency of increase in mitochondria diameter was seen with 250 microg/mL veratrine. We conclude that the alkaloid would probably act on mitochondrial membrane phospholipid configuration, which would explain the changes observed.     

Light,scanning, and transmission electron microscopy of a single population of detergent-extracted cardiac myocytes in vitro

A technique for performing light, scanning, and transverse transmission electron microscopy on cultured cells grown within a single tissue culture flask is described. Permanent light microscopy slides are obtained by removing selected portions of the plastic tissue culture vessel and mounting them on glass slides with an aqueous mounting solution. The images obtained from these slides are superior to viewing through the bottom of the flask with an inverted stage microscope. For scanning electron microscopy, selected areas are also cut from the remainder of the vessel and prepared for viewing. The final portion of the culture container is transferred and attached to a new tissue culture vessel and prepared for transmission electron microscopy using alcohol instead of acetone and propylene oxide during dehydration, infiltration, and embedding.