Elements In Biological Specimens Research Articles

X-ray fluorescence microscopy methods for biological tissues.

Synchrotron-based X-ray fluorescence microscopy is a flexible tool for identifying the distribution of trace elements in biological specimens across a broad range of sample sizes. The technique is not particularly limited by sample type and can be performed on ancient fossils, fixed or fresh tissue specimens, and in some cases even live tissue and live cells can be studied. The technique can also be expanded to provide chemical specificity to elemental maps, either at individual points of interest in a map or across a large field of view. While virtually any sample type can be characterized with X-ray fluorescence microscopy, common biological sample preparation methods (often borrowed from other fields, such as histology) can lead to unforeseen pitfalls, resulting in altered element distributions and concentrations. A general overview of sample preparation and data-acquisition methods for X-ray fluorescence microscopy is presented, along with outlining the general approach for applying this technique to a new field of investigation for prospective new users. Considerations for improving data acquisition and quality are reviewed as well as the effects of sample preparation, with a particular focus on soft tissues. The effects of common sample pretreatment steps as well as the underlying factors that govern which, and to what extent, specific elements are likely to be altered are reviewed along with common artifacts observed in X-ray fluorescence microscopy data.

Optimization of Sample Preparation Methods and SEM Imaging Conditions Enables High Resolution X-ray Mapping of Essential Elements in Biological Specimens

Optimization of Sample Preparation Methods and SEM Imaging Conditions Enables High Resolution X-ray Mapping of Essential Elements in Biological Specimens

Reproducibility of essential elements chromium, manganese, iron, zinc and selenium in spot samples, first-morning voids and 24-h collections from healthy adult men.

Evaluation of Cr, Mn, Fe, Zn and Se in humans is challenged by the potentially high within-individual variability of these elements in biological specimens, which are poorly characterised. This study aimed to evaluate their within-day, between-day and between-month variability in spot samples, first-morning voids and 24-h collections. A total of 529 spot urine samples (including eighty-eight first-morning voids and 24-h collections) were collected from eleven Chinese adult men on days 0, 1, 2, 3, 4, 30, 60 and 90 and analysed for these five elements using inductively coupled plasma-MS. Intraclass correlation coefficients (ICC) were utilised to characterise the reproducibility, and their sensitivity and specificity were analysed to assess how well a single measurement classified individuals' 3-month average exposures. Serial measurements of Zn in spot samples exhibited fair to good reproducibility (creatinine-adjusted ICC = 0·47) over five consecutive days, which became poor when the samples were gathered months apart (creatinine-adjusted ICC = 0·33). The reproducibility of Cr, Mn, Fe and Se in spot samples was poor over periods ranging from days to months (creatinine-adjusted ICC = 0·01-0·12). Two spot samples were sufficient for classifying 60 % of the men who truly had the highest (top 33 %) 3-month average Zn concentrations; for Cr, Mn, Fe and Se, however, at least three specimens were required to achieve similar sensitivities. In conclusion, urinary Cr, Mn, Fe, Zn and Se concentrations showed a strong within-individual variability, and a single measurement is not enough to efficiently characterise individuals' long-term exposures.

Multimodal correlative microscopy for in situ detection and quantification of chemical elements in biological specimens. Applications to nanotoxicology

Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological and structural information that exceeds what is possible with any single technique to answer a biological question. We propose an approach based on a multimodal correlative microscopy, via two imaging and analytical techniques: fluorescence microscopy (FM) and ion beam analysis (IBA) to investigate in vitro nanoparticles (NPs) interactions. Indeed, the explosive growth in Nanotechnology has led to their utilization in a wide range of applications from therapeutics to multimodal imaging labeling. However, the risks for adverse health effects have not been clearly established. Detecting and tracking nanomaterials in biological systems are thus challenging and essential to understand the possible NPs-induced adverse effects. Indeed, assessing in situ NPs internalization at the single cell level is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of NPs internalized per cell. The data obtained by both FM and IBA were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA provides the in situ quantification not only of exogenous elements in a single cell but also of all the other endogenous elements and the subsequent variation in their cellular homeostasis. This unique property gives access to dose-dependent response analyses and therefore new perspectives for a better insight on the effect of metal oxide NPs on cellular homeostasis.

X‐ray fluorescence elemental mapping and microscopy to follow hepatic disposition of a Gd‐based magnetic resonance imaging contrast agent

1. Spatially resolved X-ray fluorescence (XRF) spectroscopy with synchrotron radiation is a technique that allows imaging and quantification of chemical elements in biological specimens with high sensitivity. In the present study, we applied XRF techniques at a macro and micro level to carry out drug distribution studies on ex vivo models to confirm the hepatobiliary disposition of the Gd-based magnetic resonance imaging contrast agent B22956/1. 2. Gd presence was selectively quantified allowing the determination of the time dependent disappearance of the drug from blood and its hepatic accumulation in mice after administration. Elemental mapping highlighted the drug distribution differences between healthy and diseased livers. XRF microanalyses showed that in CCl(4) -induced hepatitis, B22956/1 has greatly reduced hepatic accumulation, shown as a 20-fold reduction of Gd presence. Furthermore, a significant increase of Fe presence was found in steatotic compared with healthy livers, in line with the disease features. 3. The present results show that XRF might be useful in preclinical pharmacological studies with drugs containing exogenous elements. Furthermore, quantitative and high-sensitivity elemental mapping allows simultaneous detection of chemical variation, showing pathological conditions. This approach was useful in suggesting reduced B22956/1 accumulation in steatotic livers, thus opening possible new diagnostic perspectives for this drug.

Calibration curves in atomic emission spectral analysis of biological specimens

An efficient algorithm and special software are described for plotting calibration curves in the form of polynomials of optimal degree for atomic emission spectral analysis. The operation of the software is demonstrated using software/hardware systems for monitoring the content of toxic elements in biological specimens. It is shown that using optimal calibration curves leads to a decrease in the error of the spectral analysis.

X-ray microanalysis of biological specimens by high voltage electron microscopy

For the purpose of analyzing and imaging chemical components of cells and tissues at the electron microscopic level, 3 fundamental methods are available, chemical, physical and biological. Among the physical methods, two methods qualifying and quantifying the elements in the structural components are very often employed. The first method is radioautography which can demonstrate the localization of radiolabeled compounds which were incorporated into cells and tissues after the administration of radiolabeled compounds. The second method is X-ray microanalysis which can qualitatively analyze and quantify the total amounts of elements present in cells and tissues. We have developed the two methodologies in combination with intermediate high or high voltage transmission electron microscopy (200–400 kV) and applied them to various kinds of organic and inorganic compounds present in biological materials. As for the first method, radioautography, I had already contributed a chapter to PHC (37/2). To the contrary, this review deals with another method, X-ray microanalysis, using semi-thin sections and intermediate high voltage electron microscopy developed in our laboratory. X-ray microanalysis is a useful method to qualify and quantify basic elements in biological specimens. We first quantified the end-products of histochemical reactions such as Ag in radioautographs, Ce in phosphatase reaction and Au in colloidal gold immunostaining using semithin sections and quantified the reaction products observing by intermediate high voltage transmission electron microscopy at accelerating voltages from 100 to 400 kV. The P/ B ratios of all the end products Ag, Ce and Au increased with the increase of the accelerating voltages from 100 to 400 kV. Then we analyzed various trace elements such as Zn, Ca, S and Cl which originally existed in cytoplasmic matrix or cell organelles of various cells, or such elements as Al which was absorbed into cells and tissues after oral administration, using both conventional chemical fixation and cryo-fixation followed by cryo-sectioning and freeze-drying, or freeze-substitution and dry-sectioning, or freeze-drying and dry-sectioning producing semithin sections similarly to radioautography. As the results, some trace elements which originally existed in cytoplasmic matrix or cell organelles of various cells in different organs such as Zn, Ca, S and Cl, were effectively detected. Zn was demonstrated in Paneth cell granules of mouse intestines and its P/ B ratios showed a peak at 300 kV. Ca was found in human ligaments and rat mast cells with a maximum of P/ B ratios at 350 kV. S and Cl were detected in mouse colonic goblet cells with maxima of P/ B ratios at 300 kV. On the other hand, some elements which were absorbed by experimental administration into various cells and tissues in various organs, such as Al in lysosomes of hepatocytes and uriniferous tubule cells in mice was detected with a maximum of P/ B ratios at 300 kV. From the results, it was shown that X-ray microanalysis using semi-thin sections observed by intermediate high voltage transmission electron microscopy at 300–400 kV was very useful resulting in high P/ B ratios for quantifying some trace elements in biological specimens. These methodologies should be utilized in microanalysis of various compounds and elements in various cells and tissues in various organs.

Electron Energy-Loss Spectroscopy

Solid-state scientists have many analytical techniques to choose from. But electron energy-loss spectroscopy does some things that no other technique can quite match. Electron energy-loss spectroscopy can, for example, form chemical maps of nanometre-sized regions in solid samples. It can be used to characterize interfaces buried deep in samples and to identify trace elements in biological specimens. It can even look at an individual row of atoms in a crystal and identify the type of atoms and their bonding states.

The Utility Value of High Voltage Electron Microscopy for X-Ray Microanalysis

X-ray microanalysis is a useful technique to qualify and quantify basic elements in biological specimens. article reviews the principles and techniques for applying intermediate high voltage electron microscopy to X-ray microanalysis of various elements in biological specimens developed in our laboratory since the late 20th century. We first quantified the endproducts of histochemical reactions such as Ag in radioautographs, Ce in acid phosphatase reaction and Au in colloidal goldimmunostaining using semi-thin sections by high voltage electron micros- copy at 300-400 kV. We then analyzed various trace elements such as Zn, Ca, and S, which originally existed in the cytoplasmic matrix or cell organelles This of various cells in different organs, and some absorbed elements introduced by some absorbed elements introduced by experimental administration into cells and tissues such as Al, using both conventional chemical fixation and cryo fixation followed by cryo-sectioning, freeze-drying, or freeze-substitution. As a result, we showed that the peak to background (P/B) ratios of all the elements analyzed had high P/B ratios at 300-400 kV. It was concluded that X-ray microanalysis using semi-thin sections by high voltage electron microscopy is of great utility for quantifying trace elements in biological specimens.

Electron energy-loss spectroscopy

Solid-state scientists have many analytical techniques to choose from. But electron energy-loss spectroscopy does some things that no other technique can quite match. Electron energy-loss spectroscopy can, for example, form chemical maps of nanometre-sized regions in solid samples. It can be used to characterize interfaces buried deep in samples and to identify trace elements in biological specimens. It can even look at an individual row of atoms in a crystal and identify the type of atoms and their bonding states.

QUANTITATIVE ANALYSIS OF BIOLOGICAL SAMPLES USING SYNCHROTRON RADIATION MICROPROBE

A quantitative analysis method of the trace elements in biological specimens using the synchrotron radiation microprobe is described. The synchrotron radiation X-ray fluorescence microprobe system is calibrated by the standard sample specially prepared for the microprobe analysis. The mass thickness of each measured point is determined by measuring Compton scattering intensity in 17–20 keV from the sample. In this way, concentration of the trace elements in samples can be measured correctly. The measured values of the biological standard reference material NBS-SRM-1577a are in good agreement with the certified values. The longitudinal concentration variations of some trace element in a single hair of pregnant women have been measured by this method

On the potential of combining neutron activation analysis and particle induced X-ray emission (PIXE) for micro- and microdistribution analysis of hard metals in human tissues

Tungsten carbide and cobalt are the main components of hard metal alloy while other metals such as chromium, niobium, tantalum, titanium and vanadium are sometimes added in smaller amounts. Exposure to hard metal dusts can induce a lung fibrosis with cobalt playing a major role. In order to provide information on the role that each metal may have in causing this disease, determination of the total content and the distribution of inhaled metals in lung tissue of hard metal workers is of paramount importance. However, samples such as transbronchial biopsy and bronchoalveolar lavage (BAL), often used in the medical diagnosis of pneumoconiosis, only allow for a small amount of material. This calls for sensitive and accurate analytical procedures for microdetermination and distribution of metals in pulmonary tissue and cellular material, such as macrophages. This work proposes a combination of sophisticated analytical techniques such as neutron activation analysis (NAA), currently applied to the determination of the total concentration of more than 30 elements in biological specimens, and PIXE analysis, particularly microPIXE, which has a great potential for microdistribution analysis in small biological samples. Principles and perspectives for the combined use of these techniques for the analysis of human tissue are outlined and discussed. NAA: determination of hard metals in lung tissue are carried out by neutron irradiation (2 × 10 14 neutrons cm −2 s −1) in the HFR reactor of Petten. After neutron activation, radiochemical separations of 60Co, 187W, 182Ta, 51Cr followed by computer-based high resolution gamma ray spectrometry allow the measurement of these elements in pulmonary tissues with sensitivities ranging from 10 −4 μg (Cr) to 10 −6 μg (W). PIXE: this technique is multielemental and of relatively high sensitivity (μg/g) even in small total sample masses of from 10 to 100 μg, thus allowing the analysis of parts of needle biopsies. Whereas PIXE has been successfully applied to many medical problems, its usefulness is limited in the total samples analysis for cobalt-related hard metal disease, due to the low levels of cobalt in tissue combined with severe element interferences from the generally more abundant metal, iron. Nevertheless, microPIXE, a special variety of the method scanning over the sample with a focussed ion beam of about 2 × 2 μm 2, could complement the NAA findings in total samples in the sense of achieving a microdistribution analysis of hard metals (including cobalt) in suitable thin tissue sections. The availability of specialized facilities at the JRC such as the powerful HFR reactor (Petten), the NAA laboratories (Ispra) and the microPIXE facility (Geel) could represent a European ‘reference pole’ for the study of metals in tissues of hard metal diseased subjects.

Determination of nucleic acid content of viruses by electron spectroscopic imaging and correlation averaging

Energy filtering offers the potential to form element-specific images, and thus to quantitate and map various elements in biological specimens. To assess the sensitivity and reproducibility with which we can detect phosphorus using current technology, we have performed ESI experiments in a model system comprising three well-characterized viruses - tobacco mosaic virus (TMV), LA virus of yeast, and bacteriophage T4 - that differ widely in their contents and spatial distributions of nucleic acid. Here we make a preliminary report of experiments in which the data were recorded in a Zeiss EM902 electron microscope equipped with a Henry-Castaing energy filter.An aliquot of the viral mixture was adsorbed to a thin, grid-mounted, carbon film, blotted to a thin film, rapidly frozen in liquid ethane, and transferred into the microscope in the frozen-hydrated state, where it was freeze-dried at 100°K. A SIT camera was used to record images at energy losses of 105, 120 and 150 eV respectively, with an energy window of 10eV.

Biological monitoring with atomic spectroscopy

Atomic spectroscopy holds a central role in biological monitoring. With this powerful and mature analytical tool, meaningful measurements can be made of a number of important elements in biological specimens. These measurements are useful in two general settings: (1) with toxic elements, by measurement of concentrations in excess of “expected” ranges; and (2) with essential elements, in which deficiencies may be observed. Several considerations are of major importance to effectively apply atomic spectroscopy to these measurements; (1) choice of an appropriate specimen; (2) selection of the most appropriate analytical method; (3) collection and preservation of the specimen; (4) analysis of the specimen; and (5) evaluation of the data generated. The usefulness of the various forms of atomic spectroscopy will be shown with two illustrative examples, and some evaluation will be provided of the future directions of research and upcoming issues in biological monitoring.

Contact x-ray microscopy. A new technique for imaging cellular fine structure

Contact x-ray microscopy potentially allows living, wet cells to be visualized at a resolution of up to 100 A. Furthermore, differential absorption by specific elements permits the study of the distribution of those elements in biological specimens. In contact x-ray microscopy, soft x-rays (10 A to 100 A) pass through a biological sample and expose an underlying x-ray sensitive polymer (resist), producing an image that reflects the photon absorbance within the specimen. The high penetrating power of soft x-ray enables images to be obtained from specimens up to several microns thick. In this paper, the technique is described, some of the areas currently under study are considered, and biological examples of the use of contact x-ray microscopy are given.

Quantitative X-ray microanalysis of halogen elements in biological specimens.

Standards were prepared for quantitative X-ray microanalysis of the halogen elements Cl, Br and I in sections of resin embedded biological specimens. Halogenated aromatic compounds were dissolved in resin, which subsequently was polymerized. Homogeneity and stability of the standards were determined and found to be satisfactory. A general procedure of calculation of elemental concentrations according to the continuum method is given for the case that the specimen contains appreciable concentrations of one or more rather heavy elements. It is shown that use of approximations may lead to unacceptable errors, even in the concentration ranges occurring in biological specimens. As a practical application, the concentration of bromine in the chloroplasts of the red alga Chondrus crispus was determined quantitatively. The inner cells contained more bromine than the epidermal cells: in the chloroplasts of the inner cells bromine concentrations of about 6% could be demonstrated.

A half automated, non time consuming radiochemical separation scheme for determination of 25 trace elements in biological specimens

A radiochemical separation scheme for the determination of 25 neutron activated trace elements in biological soft tissue is represented. The scheme includes wet ashing and 16 different organic and inorganic ion exchangers. The procedure is semiautomatized and a description for manufactoring the non commercial available exchangers is given. Precision of the scheme is determined for each single element as well as for a mixture of all elements together.

Line-focusing x-ray monochromator for the analysis of trace elements in biological specimens.

A line-focusing x-ray monochromator has been designed and developed to be used with an energy dispersive detection system for the quantitative analysis of trace elements in biological specimens. This instrument uses a cylindrically curved Johansson-type crystal to monochromatize and focus the excitation radiation into a line 0.5 mm wide and 40 mm long. The excitation radiation chosen for these experiments was Mo Kalpha and the total intensity of the line-focused beam was estimated to be 2 X 10(7) cps. Because of the narrow width and high power density of the excitation between beam, small tissue specimens 1 X 1 X 12 mm in size along with a single strand of hair were analyzed for trace elements from Al through Sr. For the transition elements, concentrations of a few parts per million were easily detected with this analyzer.

An Energy Dispersive System for the Analysis of Trace Elements in Human Blood Serum

AbstractA computerized systetn, consisting of a fluorescence x-ray source (Philips), an Si (Li) detector (Ortec), and a mini Computer (Xerox) is currentiy in operation at The University of Texas M. D. Anderson Hospital and Tumor Institute, Experimentel Pathology Section, for the analysis of trace elements in biological specimens. The elements of interest are Fe, Cu, and Zn for possible significance in the detection and study of cancer. The detection system is being used for comparatively routine analysis of these elements, which are present in blood serum in the ppM range. With appropriate sample preparation techniques, using 1 ml of serum, the limit of detectability for these elements is estimated to be 100 ppB (1 in 107). Further refinement is possible, and research in this direction continues.