Development and comparison of the methods for quantitative electron probe X‐ray microanalysis analysis of thin specimens and their application to biological material

In recent years, there has been a return to the use of electron probe X‐ray microanalysis for biological studies but this has occurred at a time when the Hall programme which acted as the mainstay for biological microanalysis is no longer easily available. Commercial quantitative routines rely on the Cliff‐Lorimer method that was originally developed for materials science applications. Here, the development of these two main routines for obtaining quantitative data from thin specimens is outlined and the limitations that are likely to be met when the Cliff‐Lorimer routine is applied to biological specimens is discussed. The effects of specimen preparation on element content is briefly summarized and the problems encountered when using quantitative analysis on resin‐embedded materials emphasized.     

The use of a phi(rhoz) model for simultaneous determination of composition and thickness in analytical transmission electron microscopy

X-ray microanalysis using transmission electron microscopy (TEM) offers the possibility to perform quantitative analysis with high spatial resolution. Disadvantages are its low accuracy and the problem of preparing very thin specimens and the thin film standards, needed for the analysis and calibration. To calculate composition from the measured X-ray intensities, a peak-ratio method is usually applied, based on the thin film method by Cliff and Lorimer (Proceedings of the Fifth European Congress on Electron Microscopy, 1972, p. 140). We however, applied an entirely different approach, calculating the composition using a full matrix correction method based upon a phi(rhoz) matrix correction model as they are commonly used in EPMA measurements up to 40 kV. The validity of the model under TEM conditions was checked by performing bulk analyses on AlNi and AlTi samples and thin film analyses on an AlNi TEM specimen. In principle, both thin and thick specimens as well as light elements can be analysed this way. No major changes to the TEM set-up or simplifications to the model are needed, only an accurate beam current meter is required.     

Cells on biomaterials – some aspects of elemental analysis by means of electron probes

Electron probe X‐ray microanalysis enables concomitant observation of specimens and analysis of their elemental composition. The method is attractive for engineers developing tissue‐compatible biomaterials. Either changes in element composition of cells or biomaterial can be defined according to well‐established preparation and quantification procedures. However, the qualitative and quantitative elemental analysis appears more complicated when cells or thin tissue sections are deposited on biomaterials. X‐ray spectra generated at the cell/tissue–biomaterial interface are modelled using a Monte Carlo simulation of a cell deposited on borosilicate glass. Enhanced electron backscattering from borosilicate glass was noted until the thickness of the biological layer deposited on the substrate reached 1.25 μm. It resulted in significant increase in X‐ray intensities typical for the elements present in the cellular part. In this case, the mean atomic number value of the biomaterial determines the strength of this effect. When elements are present in the cells only, the positive linear relationship appears between X‐ray intensities and cell thickness. Then, spatial dimensions of X‐ray emission for the particular elements are exclusively in the range of the biological part and the intensities of X‐rays become constant. When the elements are present in both the cell and the biomaterial, X‐ray intensities are registered for the biological part and the substrate simultaneously leading to a negative linear relationship of X‐ray intensities in the function of cell thickness. In the case of the analysis of an element typical for the biomaterial, strong decrease in X‐ray emission is observed in the function of cell thickness as the effect of X‐ray absorption and the limited excitation range to biological part rather than to the substrate. Correction procedures for calculations of element concentrations in thin films and coatings deposited on substrates are well established in materials science, but little is known about factors that have to be taken into account to accurately quantify bioelements in thin and semi‐thick biological samples. Thus thorough tests of currently available quantification procedures are required to verify their applicability to cells or tissues deposited on the biomaterials.     

New pathways for improved quantification of energy‐dispersive X‐ray spectra of semiconductors with multiple X‐ray lines from thin foils investigated in transmission electron microscopy

Theoretical approaches to quantify the chemical composition of bulk and thin‐layer specimens using energy‐dispersive X‐ray spectroscopy in a transmission electron microscope are compared to experiments investigating (In)GaAs and Si(Ge) semiconductors. Absorption correctors can be improved by varying the take‐off angle to determine the depth of features within the foil or the samples thickness, or by definition of effective k‐factors that can be obtained from plots of k‐factors versus foil thickness or, preferably, versus the K/L intensity ratio for a suitable element. The latter procedure yields plots of self‐consistent absorption corrections that can be used to determine the chemical composition, iteratively for SiGe using a set of calibration curves or directly from a single calibration curve for InGaAs, for single X‐ray spectra without knowledge of sample thickness, density or mass absorption coefficients.     

Transmission electron microscopic X-ray quantitative analysis of human dentin at 200 kV accelerating voltage

Qualitative and quantitative x-ray energy dispersive spectroscopy is now used successfully to analyze many features and processes in inorganic samples. When applied to inorganic samples, however, the results are often less satisfactory due to problems of preparation of organic samples, difficulty of measuring x-rays from organic samples, damage of the sample by the electron beam, and other practical problems. In the present study we used a high voltage transmission electron microscope equipped with an energy dispersive x-ray spectrometer to examine accurate quantitative standardless analysis of thin sections of an organic sample, human dentin. Based on our experiments we found the important parameters for quantitative analysis were sample thickness and appropriate choice of model sample. Further, we show that the method of Cliff and Lorimer can be used with biological samples at 200 kV, and we show that quantitative analysis of human dentin can be carried out at 200 kV. Finally, we show that areas of human dentin can be differentiated by their morphological characteristics and x-ray analyses obtained in the transmission electron microscope.     

Quantitative imaging of Candida utilis and its organelles by soft X‐ray Nano‐CT

Soft X‐ray microscopy has excellent characteristics for imaging cells and subcellular structures. In this paper, the yeast strain, Candida utilis, was imaged by soft X‐ray microscopy and three‐dimensional volumes were reconstructed with the SART‐TV method. We performed segmentation on the reconstruction in three dimensions and identified several types of subcellular architecture within the specimen cells based on their linear absorption coefficient (LAC) values. Organelles can be identified by the correlation between the soft X‐ray LAC values and the subcellular architectures. Quantitative analyses of the volume ratio of organelles to whole cell in different phases were also carried out according to the three‐dimensional datasets. With such excellent features, soft X‐ray imaging has a great influence in the field of biological cellular and subcellular research.     

Is Scanning Electron Microscopy/Energy Dispersive X‐ray Spectrometry (SEM/EDS) Quantitative?

Scanning electron microscopy/energy dispersive X‐ray spectrometry (SEM/EDS) is a widely applied elemental microanalysis method capable of identifying and quantifying all elements in the periodic table except H, He, and Li. By following the “k‐ratio” (unknown/standard) measurement protocol development for electron‐excited wavelength dispersive spectrometry (WDS), SEM/EDS can achieve accuracy and precision equivalent to WDS and at substantially lower electron dose, even when severe X‐ray peak overlaps occur, provided sufficient counts are recorded. Achieving this level of performance is now much more practical with the advent of the high‐throughput silicon drift detector energy dispersive X‐ray spectrometer (SDD‐EDS). However, three measurement issues continue to diminish the impact of SEM/EDS: (1) In the qualitative analysis (i.e., element identification) that must precede quantitative analysis, at least some current and many legacy software systems are vulnerable to occasional misidentification of major constituent peaks, with the frequency of misidentifications rising significantly for minor and trace constituents. (2) The use of standardless analysis, which is subject to much broader systematic errors, leads to quantitative results that, while useful, do not have sufficient accuracy to solve critical problems, e.g. determining the formula of a compound. (3) EDS spectrometers have such a large volume of acceptance that apparently credible spectra can be obtained from specimens with complex topography that introduce uncontrolled geometric factors that modify X‐ray generation and propagation, resulting in very large systematic errors, often a factor of ten or more. SCANNING 35: 141‐168, 2013. ¹ Published 2012 Wiley Periodicals, Inc.     

Contact X‐ray microscopy of living cells by using LiF crystal as imaging detector

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single‐shot soft X‐ray contact microscopy is presented. High resolved X‐ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X‐ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid‐state X‐ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X‐ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X‐ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high‐quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.     

A vacuum-compatible wet-specimen chamber for compact X-ray microscopy

Soft X‐ray microscopy is a powerful tool for investigations of, for example, polymers or soils in their natural liquid environment. This requires a wet‐specimen chamber. Compact X‐ray microscopy allows the horizontal mounting of such samples, thereby reducing the influence of gravitational forces. We have developed a wet‐specimen chamber for such compact X‐ray microscope. The chamber is vacuum compatible, which reduces the exposure time. The vacuum sealing is achieved by a combination of mechanical sealing and sealing by bio‐compatible glue. With the wet‐specimen chamber the specimens can be kept in an aqueous environment in a vacuum of 10⁻⁴ mbar for several hours. Imaging of lipid droplets in water demonstrates the function of the wet‐specimen chamber.     

The secondary fluorescence correction for X-ray microanalysis in the analytical electron microscope

A general formulation for the secondary fluorescence correction is presented. It is intended to give an intuitive appreciation for the various factors that influence the magnitude of the secondary fluorescence correction, the specimen geometry in particular, and to serve as a starting point for the derivation of quantitative correction formulae. This formulation is primarily intended for the X-ray microanalysis of electron-transparent specimens in the analytical electron microscope (AEM). The fluoresced intensity, I^Y_X, is expressed relative to the primary intensity of the fluorescing element, I_Y, rather than to that of the fluoresced element, I_X, as has been customary for microanalysis. The importance of this choice of I_Y as a reference intensity for the electron-transparent specimens examined in the AEM is discussed. The various factors entering the secondary fluorescence correction are grouped into three factors, representing the dependencies of the correction on specimen composition, X-ray fluorescence probability and specimen geometry. In principle, an additional factor should be appended to account for the difference in detection efficiencies of the fluoresced and fluorescing X-rays; however, this factor is shown to be within a few per cent of unity for practical applications of the secondary fluorescence correction. The absorption of secondary X-rays leaving the specimen en route to the detector is also accounted for through a single parameter. In the limit that the absorption of secondary X-rays is negligible, the geometric factor has the simple physical interpretation as the fractional solid angle subtended by the fluoresced volume from the perspective of the analysed volume. Studies of secondary fluorescence in the published literature are compared with this physical interpretation. It is shown to be qualitatively consistent with Reed's expression for secondary fluorescence in the electron probe microanalyser and with the specimen-thickness dependence of the Nockolds expression for the parallel-sided thin foil. This interpretation is also used to show that the ‘sec α’ dependence on specimen tilt in the latter expression is erroneous and should be omitted. The extent to which extrapolation methods can be used to correct for secondary fluorescence is also discussed. The notion that extrapolation methods, by themselves, can be used to correct for secondary fluorescence is refuted. However, extrapolation methods greatly facilitate secondary fluorescence correction for wedge-shaped specimens when used in conjunction with correction formulae.     

Size-selective colloidal-gold localization in transmission X-ray microscopy

Colloidal gold is a useful marker for functional‐imaging experiments in transmission X‐ray microscopy. Due to the low contrast of gold particles with small diameters it is necessary to develop a powerful algorithm to localize the single gold particles. The presented image‐analysis algorithm for identifying colloidal gold particles is based on the combination of a threshold with respect to the local absorption and shape discrimination, realized by fitting a Gaussian profile to the identified regions of interest. The shape discrimination provides the possibility of size‐selective identification and localization of single colloidal gold particles down to a diameter of 50 nm. The image‐analysis algorithm, therefore, has potential for localization studies of several proteins simultaneously and for localization of fiducial markers in X‐ray tomography.     

A technique for the examination of polar ice using the scanning electron microscope

The microstructure and location of impurities in polar ice are of great relevance to ice core studies. We describe a reliable method to examine ice in the scanning electron microscope (SEM). Specimens were cut in a cold room and could have their surfaces altered by sublimation either before (pre‐etching) or after (etching) introduction to the cryo‐chamber of the SEM. Pre‐etching was used to smooth surfaces, whilst etching stripped away layers from the specimen surface, aiding the location of particles in situ, and allowing embedded structures to be revealed. X‐ray analysis was used to determine the composition of localized impurities, which in some cases had been concentrated on the surface by etching. Examining uncoated surfaces was found to be advantageous and did not detract from qualitative X‐ray analysis. Imaging uncoated was performed at low accelerating voltages and probe currents to avoid problems of surface charging.     

Application of the parametric bootstrap method to determine statistical errors in quantitative X-ray microanalysis of thin films

We applied the parametric bootstrap to the X‐ray microanalysis of Si‐Ge binary alloys, in order to assess the dependence of the Ge concentrations and the local film thickness, obtained by using previously described Monte Carlo methods, on the precision of the measured intensities. We show how it is possible by this method to determine the statistical errors associated with the quantitative analysis performed in sample regions of different composition and thickness, but by conducting only one measurement. We recommend the use of the bootstrap for a broad range of applications for quantitative microanalysis to estimate the precision of the final results and to compare the performances of different methods to each other. Finally, we exploited a test based on bootstrap confidence intervals to ascertain if, for given X‐ray intensities, different values of the estimated composition in two points of the sample are indicative of an actual lack of homogeneity.     

Quantitative x‐ray microanalysis of model biological samples in the SEM using remote standards and the XPP analytical model

It is shown that accurate x‐ray microanalysis of frozen‐hydrated and dry organic compounds, such as model biological samples, is possible with a silicon drift detector in combination with XPP (exponential model of Pouchou and Pichoir matrix correction) software using ‘remote standards’. This type of analysis is also referred to as ‘standardless analysis’. Analyses from selected areas or elemental images (maps) were identical. Improvements in x‐ray microanalytical hardware and software, together with developments in cryotechniques, have made the quantitative analysis of cryoplaned frozen‐hydrated biological samples in the scanning electron microscope a much simpler procedure. The increased effectiveness of pulse pile‐up rejection renders the analysis of Na, with ultrathin window detectors, in the presence of very high concentrations of O, from ice, more accurate. The accurate analysis of Ca (2 mmol kg^?1) in the presence of high concentrations of K is possible. Careful sublimation of surface frost from frozen‐hydrated samples resulted in a small increase in analysed elemental concentrations. A more prolonged sublimation from the same resurfaced sample and other similar samples resulted in higher element concentrations.     

Morphological properties of Ag₂SeTe nano thin films prepared by thermal evaporation

Semiconducting silver selenide telluride (Ag₂SeTe) thin films were prepared with different thicknesses onto glass substrates at room temperature using thermal evaporation technique. The structural properties were determined as a function of thickness by X‐ray diffraction exhibiting no preferential orientation along any plane; however, the films are found to have peaks corresponding to mixed phase. The morphology of these films was studied using scanning electron microscope and atomic force microscopy respectively, and is reported. The morphological properties are found to be very sensitive to the thin film thickness. The composition of the films is also estimated using energy dispersive analysis using X‐rays and are also reported.     

Phase‐contrast hard X‐ray microscopy using synchrotron radiation for the properties of skeletal muscle in mouse hind limbs

Radiation beam interface contrast X‐ray microscopy provides resolution of a few dozen nanometers from fixed whole muscle biopsies, allowing better reconstruction of the microstructure of the muscle than is currently possible with classic histological techniques. Fixed soleus muscle biopsies have been evaluated from the walk‐in mouse model using phase‐contrast X‐ray microscopy, and results presented that corroborate the accuracy of the method used, and its potential for application in physiotherapy and occupational therapy studies. We believe that this method will enhance existing morphometric methods of analysis, leading to accurate reconstruction of other thick specimens that would otherwise require thin sectioning and reconstruction through deconvolution algorithms.     

Detection of lead in Zea mays by dual‐energy X‐ray microtomography at the SYRMEP beamline of the ELETTRA synchrotron and by atomic absorption spectroscopy

This study is related to the application of the X‐ray dual‐energy microradiography technique together with the atomic absorption spectroscopy (AAS) for the detection of lead on Zea mays stem, ear, root, and leaf samples. To highlight the places with lead intake, the planar radiographs taken with monochromatic X‐ray radiation in absorption regime with photon energy below and above the absorption edge of a given chemical element, respectively, are analyzed and processed. To recognize the biological structures involved in the intake, the dual‐energy images with the lead signal have been compared with the optical images of the same Z. mays stem. The ear, stem, root, and leaf samples have also been analyzed with the AAS technique to measure the exact amount of the hyperaccumulated lead. The AAS measurement revealed that the highest intake occurred in the roots while the lowest in the maize ears and in the leaf. It seems there is a particular mechanism that protects the seeds and the leaves in the intake process. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc. This article was published online on 1 December 2009. An error was subsequently identified. This notice is included in the online and print version to indicate that both have been corrected 19 February 2010.     

Exploring the use of soft X‐ray microscopy for imaging subcellular structures of the inner ear1

The soft X‐ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X‐ray microscopy provides high‐resolution visualization of hydrated, non‐embedded and non‐sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X‐ray micrographs of structures isolated from the guinea‐pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X‐ray images of the acellular tectorial membrane and thin two‐layered Reissner's membrane display a level of resolution comparable to low‐power electron microscopy.     

Optical characteristics of thin film coating and measurement of its thickness

Theoretical analysis of the optical characteristics of thin film coatings was carried out. The relations of refractive index, phase change and film thickness were studied. As a result, a new optical method of measuring thickness of thin metallic coatings was developed. Further, the technique can also be used to find the refractive index of the thin film and the phase change of the light beam owing to being reflected from the coating. It is shown that the new method is very accurate for measuring the thin coating thickness. A specimen coating was tested with a polarised laser beam and the measured phase change falls between the reasonable range of 0 and π.