DETERMINATION OF PICOMOLE CONCENTRATIONS OF ALUMINUM (III) IN HUMAN SALIVA AND URINE BY A LUMINOL-CARBOXYMETHYL CHITOSAN CHEMILUMINESCENCE SYSTEM

A luminol–carboxymethyl chitosan (CMCS) system was established using flow injection chemiluminescence (CL) based on the enhancing effect of CMCS on the luminol–dissolved oxygen reaction. The CL intensity was linear with the CMCS concentration ranging from 0.01–30.0 µM. Al(III) was shown to quench the CL of the luminol–CMCS reaction, and the decrease of CL intensity was linear with the logarithm of Al(III) concentration over the range from 10–1000 pM with a detection limit of 3.5 pM (3σ). At a flow rate of 2.0 mL min^?1, the analysis was performed within 30 s. The proposed CL method was successfully applied to the determination of picomole levels Al(III) in human saliva and urine after oral intake of two aluminum hydroxide tablets, with recoveries from 90.6–108.7% and relative standard deviations <3% (n = 5). The results indicated that the excreted Al(III) in saliva and urine reached its maximum values at 3 hr and 2 hr, and the total excretive ratio were 1.24 × 10^?3% and 3.45% in 6 hr and 12 hr, respectively. The elimination rate constant k and the half–life time t _1/2 in human saliva and urine were 0.3747 hr^?1, 1.8495 hr, 0.7132 hr^?1, and 0.9717 hr, respectively. The possible CL mechanism of the luminol–CMCS–Al(III) reaction is discussed.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. I. Glutaraldehyde, osmium tetroxide, Araldite methods

The loss of ¹⁴C ethanolamine- and ³H choline-labelled phospholipids from rat liver during tissue preparation for electron microscopy has been examined. Column and thin-layer chromatography combined with double-label scintillation spectrometry were used to analyse the radioactive phospholipid content of the livers of rats injected simultaneously with ¹⁴C aminoethanol and ³H choline chloride. After 4 h (in vivo) the ¹⁴C and ³H labels were mainly incorporated into phosphatidyl ethanolamine and phosphatidyl choline respectively but some ¹⁴C label had been incorporated into phosphatidyl choline. Chopped rat liver was fixed in glutaraldehyde or osmium tetroxide or both sequentially and tissues were dehydrated in ethanol and embedded in Araldite. In each procedure examined the choline label proved more labile than the ethanolamine. After glutaraldehyde fixation alone complete loss of phosphatidyl choline occurred and half of the phosphatidyl ethanolamine was also lost. Following osmium tetroxide fixation negligible loss of either phosphatide occurred. In terms of phospholipid retention, no advantage was gained by glutaraldehyde fixation prior to osmium tetroxide fixation. The results show that both ethanols and embedding monomers are potent phospholipid solvents. The data also suggests that EM autoradiography of these two phosphatides may be carried out with reasonable confidence although it must be pointed out that a high degree of retention does not necessarily imply retention in situ.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

Quantitative image analysis of finite porous media

Software for the reconstruction of branch-node charts from serial sections is tested with a simple cubic lattice, and is applied to determine the genus per unit volume (specific genus) of a Berea sandstone sample from seventy-eight serial sections. The genus is found analytically for the cubic lattice as a function of depth, cross-section and volume. These results enable us to draw conclusions about the relative importance of depth and cross-section and provide useful information on how to treat finite sample data to obtain the specific genus of an infinite homogeneous porous medium. It is shown that it is more important to have a deep sample (many serial sections) than a sample of large cross-section. For samples which are too small in the direction of sectioning, the genus and the rate of change of genus with volume are too low because some of the large loops are not completed within the sample boundaries. For the Berea sample, this ‘shallow-sample depth’ is about 2·2 grain diameters (forty-five serial sections). Only data points at depths in excess of this value are used in determining the genus per unit volume. The slope of the genus versus volume curve is the better predictor of the genus per unit volume of the unbounded medium, of which the sample is a small part, than the value of genus/volume of the sample. For samples of finite cross-section, the G_min and G_max versus volume curves are divergent and they only become essentially parallel when the dimensions in the plane of sectioning are large multiples of the grain size. It is shown that it is reasonable to assume that the arithmetic mean slope found for a small cross-sectional area is a good estimate of the common slope that would be attained at high cross-sectional areas. Therefore, samples of small cross-section can be used, resulting in a reduction in the amount of data to be processed and an enhancement of the resolution of small features. The Berea sample data, beyond the ‘shallow-sample’ region, show the same qualitative features as predicted analytically for the cubic lattice. The ratio of the slopes of the G_max and G_min curves is 1·22 and the genus per unit volume from the mean slope is 52·6 × 10^?8 μm^?3.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Application of morphometrical methods in studies of the mandibular condyle

Two mandibular condyles of a growing monkey (Macaca fascicularis) were used for developing and testing a morphometrical model system suitable for generating quantitative data about the structure of and the growth activities in the articulating surface layers. The animal received ³H-proline (1 mCi/kg body weight) 24 h and ³H-thymidine (0.5 mCi/kg body weight) 3 h prior to killing. Using standardized conditions, 1–2 μm thick histological sections were processed for autoradiography. The morphometrical analysis was carried out using light microscopy at two levels of magnification. Sampling of tissue fields was performed at twenty-four (level I) and twelve (level II) different sites, respectively. These sites were distributed systematically in the anteroposterior and lateromedial dimension of the cartilaginous condylar covering. Using a multivariate analysis of variance, a parameter comparison revealed significant differences between single sampling sites in the anteroposterior as well as in the lateromedial direction, suggesting that the articulating condylar surface is inhomogeneous with respect to both structures and growth activities. The sampling mode eventually arrived at in this study was shown to be sufficient for the recording of gradients within the condylar surface layers. The model system presented permits simultaneous quantitative characterization of structure and growth activities such as mitotic cell division and extracellular matrix production of the condylar cartilage including the zone of its replacement by bone.     

Combining Ar ion milling with FIB lift-out techniques to prepare high quality site-specific TEM samples

Focused ion beam (FIB) techniques can prepare site‐specific transmission electron microscopy (TEM) cross‐section samples very quickly but they suffer from beam damage by the high energy Ga⁺ ion beam. An amorphous layer about 20–30 nm thick on each side of the TEM lamella and the supporting carbon film makes FIB‐prepared samples inferior to the traditional Ar⁺ thinned samples for some investigations such as high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). We have developed techniques to combine broad argon ion milling with focused ion beam lift‐out methods to prepare high‐quality site‐specific TEM cross‐section samples. Site‐specific TEM cross‐sections were prepared by FIB and lifted out using a Narishige micromanipulator onto a half copper‐grid coated with carbon film. Pt deposition by FIB was used to bond the lamellae to the Cu grid, then the coating carbon film was removed and the sample on the bare Cu grid was polished by the usual broad beam Ar⁺ milling. By doing so, the thickness of the surface amorphous layers is reduced substantially and the sample quality for TEM observation is as good as the traditional Ar⁺ milled samples.     

An X-ray microanalytical examination of precipitation methods for the ultrastructural localization of potassium in plant tissue: I. Cobaltinitrite

In vitro and in vivo studies were carried out to assess the feasibility of adapting the cobaltinitrite method for the ultrastructural localization of K⁺ in plant cells. The relatively low mobility of the cobaltinitrite ion and the solubility of the precipitation product resulted in the formation of only a few large deposits in each cell. The addition of Ag⁺ at the time of precipitation was found to increase the number of small deposits retained in sections. However, cobaltinitrite reagents frequently caused plasmolysis and poor cytoplasmic preservation. Precipitation during freeze-substitution was also attempted. It was concluded that precipitation of K⁺ using cobaltinitrite has only limited use in low magnification electron microscopy in conjunction with light microscopy.     

Freeze-substitution and the preservation of diffusible ions

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (Tl⁺) of Tl⁺-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray micro-analytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K⁺ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used; so far, in the best case, about 67% of the cellular K⁺ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.     

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.     

Radiation damage to lyophilized mineral solutions during electron probe analysis: quantitative study of chlorine loss as a function of beam current-density and sample mass-thickness

The quantitative effects of beam current-density and sample mass-thickness on the loss of chlorine which occurs from lyophilized solutes of micro-droplets of mineral salt solutions irradiated in an electron probe analyser were studied. Results are reported for chlorine loss from lyophilized deposits with mass-thickness varying between 5 and 50 mg mm^?2 for NaCl salts and 5 and 80 mg mm^?2 for KCl salts. Electron accelerating voltage was kept constant at 15 kV. The range of beam current-density (I/S, current/sample surface area) was from 0.1 to 1.5 A mm^?2. Samples were irradiated for 1200 s. The results show that under some conditions there is a period of stable chlorine signal before chlorine loss occurs. This is observed between 0.1 and 1 A mm^?2, for a period which can last several hundred seconds depending on beam current-density and sample mass-thickness. For each value of I/S, however, no stable chlorine signal can be observed for samples whose mass-thickness exceeds a value negatively correlated with I/S. The curves of decrease of characteristic chlorine X-ray signal (expressed as per cent of count rate in the initial counting interval) versus irradiation time can be fitted by the sum of two exponentials with half lives T₁ and T₂. In NaCl, T₁ and T₂ values are highly correlated with I/S but not with mass-thickness. In KCl, T₁ is correlated only with mass-thickness and T₂ only with I/S. Mixing plasma with mineral solutions prevents chlorine loss.     

Water loss at normal enamel histological points during air drying at room temperature

This in vitro study aimed to quantify water loss at histological points in ground sections of normal enamel during air drying at room temperature (25°C) and relative humidity of 50%. From each of 10 ground sections of erupted permanent human normal enamel, three histological points (n = 30) located at 100, 300 and 500 μm from enamel surface and along a transversal following prisms paths were characterized regarding the mineral, organic and water volumes. Water loss during air drying was from 0 to 48 h. Drying occurred with both falling and constant‐drying rates, and drying stabilization times (T_eq) ranged from 0.5 to 11 h with a mean 0.26 (±0.12)% weight loss. In some samples (n = 5; 15 points), T_eq increased as a function of the distance from the enamel surface, and drying occurred at an apparent diffusion rate of 3.47 × 10^?8 cm² s^?1. Our data provide evidence of air drying resulting in air replacing enamel's loosely bound water in prisms sheaths following a unidirectional water diffusion rate of 3.47 × 10^?8 cm² s^?1 (from the original enamel surface inward), not necessarily resulting in water evaporating directly into air, with important implications for transport processes and optical and mechanical properties.     

Noninvasive Quantification of Cutaneous Oedema in Patch Test Reactions by Fiber Optic Near-Infrared Fourier Transform Raman Spectroscopy

The major aim of the present study was to quantitatively differentiate various degrees of positive patch test reactions by estimating relative water content noninvasively using fiber optic near-infrared Fourier transform (NIR-FT) Raman spectroscopy. The specific intent was to examine its technical applicability and reproducibility in reading patch tests at 48 and 72 hr. Instrumental measurements at 63 patch test sites were compared visually in 19 patients with suspected allergic contact dermatitis. Raman spectra were measured in vivo in skin reacting to patches on the back using a fiber optic technique. Negative (–), doubtful (?+) and positive patch test reactions (+; ++/+++) could be separated by means of measured differences in absolute values of the 3250 cm^–1 peak (ratio I₃₂₅₀/I₂₉₄₀): at 48 hr the mean increases were 0.31(–), 0.40(?+); 0.45(+); 0.69(++/+++) and at 72 hr they were 0.31(–); 0.34(?+); 0.42(+); 0.60(++/+++). At 48 hr significant differences (p<0.05) were shown between all reactions, but not between negative and doubtful reactions at 72 hr. These findings indicate that cutaneous oedema in patch test reactions can be noninvasively quantified based on positive patch test reactions by NIR-FT Raman spectroscopy, with continuous data grading of reaction intensity suitable for clinical studies at 48 and 72 hr. We also demonstrated good technical reproducibility of patch test reactions evaluation by NIR-FT Raman spectroscopy at 48 and 72 hr, showing that this method can be used to monitor the dynamics of these reactions and display the results spectroscopically.     

How effective is ‘high-speed’ autoradiography?

A number of methods have recently been described for enhancing silver grain production in light microscope autoradiography by impregnating the emulsion with a 2,5-diphenyloxazole-based fluor system. After extensive testing of these methods it was found that none gave a significant increase in silver grain count. In the course of this work a number of observations were made which seem to account at least partially for these failures, and which question the feasibility of this type of fluorography.

• 1 Tetrahymena nuclei, pulse labelled with ³H-thymidine, contain sufficient radioactivity to produce useful autoradiographs after 2 h exposure without fluorographic enhancement.
• 2 Although fluorographic autoradiography of this type requires that the fluor penetrate the photographic emulsion, direct evidence was obtained that such penetration does not occur. Thus it is unlikely that true fluorographic enhancement occurs with methods based on 2,5-diphenyloxazole impregnation of the emulsion. (This is in contrast to the widely used gel fluorography techniques in which it is the gel rather than the photographic emulsion which is impregnated with fluor.)
• 3 In DNA fibre autoradiography, it is possible to obtain artefactual arrays of silver grains which closely resemble those expected of replicating DNA. This phenomenon alone, however, does not explain a recent report of successful DNA fibre autoradiography after an exposure of only 3 days. Calculations based on the maximum dpm per length of labelled DNA raise further questions concerning this result.

     

Image-based determination of chord lengths in air-dried lungs

An image-based method was developed in order to determine chord lengths in sections of dog and sheep lungs air-dried at 25 cm H₂O transpulmonary pressure. To facilitate image processing, optical contrast in the sections was optimized with respect to section thickness, stain type, stain concentration, staining temperature, staining time and clearing method. Digital processing of images used standard procedures, e.g. thresholding, dilation and thinning, as well as algorithms written to subtract background, delete spots and measure chord lengths. Correlation of image-based vs. manual determination of mean chord length in 17 sections from a sheep lung, stained for optimal contrast yielded an R² of 0.82 (P < 0.0001). For 95 sections from three dog lungs, stained with lower contrast, R² was 0.65 (P < 0.0001). Weaker correlations were observed between image-based and manual determinations of the standard deviation, the geometric standard deviation, and the 95th percentile of chord lengths (P < 0.05). The results show that image-based stereology of inflated air-dried lungs can provide valid measures of mean chord length and other statistics of chord length distribution.     

The use of quadrats and test systems in stereology,including magnification corrections

At present a model-free, design-based theory of unbiased estimation, and a model-based one of linear unbiased estimation of minimum variance, are available for stereology. The main developments rest upon the nested scheme {section (quadrat)}, whence the raw data are expressible in terms of area, length and number. The main aim of this paper is to complete the available model-based theory by introducing the step in which sections are analysed by point-counting via coherent test systems (CTSs). Using this development, the stereologist should be able to handle raw point and intersection counts optimally, in order to find the best estimator of a ratio R in a given specimen in a wide range of circumstances. The latter include, for instance, the use of different CTSs on different sections and of double CTSs on each section, as well as the case—(not uncommon in electron microscopy)—in which different sections from the same sample are observed at slightly different magnifications but analysed by quadrats (via automatic or semi-automatic image analysers, for instance), or CTSs of fixed sizes. The main conclusion pertaining to the latter case is that the estimators obtained via section-wise magnification corrections are in general superior to those corrected via a global, average magnification. In order to illustrate the methodology, a synthetic numerical example, and a real one, are given.     

A source of broad electron beams with a self-heated hollow cathode for plasma nitriding of stainless steel

The principle of operation and characteristics of a broad electron beam source based on the discharge with a self-heated hollow cathode and widened anode part are described. The source is intended for the ion nitriding of metals in the electron beam plasma. The influence of the current density (1–7 mA/cm²) and ion energy (0.1–0.3 keV) on the nitriding rate of the 12X18H10T austenitic stainless steel is studied. It is shown that the maximal nitriding rate is reached by the combining of the minimal bias voltage across the samples (100 V) and maximal ion current density, which ensures the dynamic oxide layer sputtering on the sample surface. The electron source, in which electrons are extracted through a stabilizing grid in the direction normal to the axis of the hollow cathode, ensures the radially divergent electron beam formation with a 700-cm² initial cross section, a current of up to 30 A, and initial electron energy of 0.1–0.5 keV. The source stably operates at nitrogen-argon mixture pressures of up to 3 Pa.     

Measuring error and sampling variation in stereology: comparison of the efficiency of various methods for planar image analysis

An evaluation is made of the relative efficiency (precision of the final estimate per unit time of measurement on a given set of sections) of different methods for planar analysis aimed at estimating aggregate, overall stereological parameters (such as V_v, S_v). The methods tested are point-counting with different densities of test points (4 ≤ P_T ≤ 900 per picture), semiautomatic computer image analysis with MOP and automatic image analysis with Quantimet, for obtaining V_v and S_v estimates. One biological sample as well as three synthetic model structures with known coefficients of variation between sections are used. The standard error of an estimate is mainly determined by the coefficient of variation between sampling units (= sections in the present paper) so that measuring each sample unit with a very high precision is not necessary. Automatic image analysis and point-counting with a 100-point grid were the most efficient methods for reducing the relative standard errors of the V_v and S_v estimates to equivalent levels in the synthetic models. Using a 64-point grid was as precise, and about 11 times faster than using a tracing device for obtaining the estimate of V_v in the biological sample.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10³ e/nm²) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10¹⁰ e/nm²) is required resulting in mass loss that may be attributable to ‘knock-on’ radiation damage.