Trace element mapping of a single cell using a hard x‐ray nanobeam focused by a Kirkpatrick‐Baez mirror system

To visualize the distributions of trace elements in biological samples such as tissues and cells at high spatial resolution, we developed a scanning x‐ray fluorescence microscope (SXFM) at SPring‐8, using a Kirkpatrick‐Baez mirror optics that enables achromatic and highly efficient focusing. To evaluate performance regarding its application to biological samples, the SXFM was used at x‐ray energy of 15 keV to observe NIH/3T3 cells in which adenosine triphosphate (ATP) synthase β (specifically localized at the mitochondria) were labeled with gold colloidal particles. Various elemental distributions were visualized at the single‐cell level, including those for P, S, Cl, Ca, Fe, Cu, Zn and Au, and we obtained high‐resolution elemental distribution maps by magnifying the labeled single mitochondrion. Maximum spatial resolution achieved in the experiments was sub‐100 nm. Copyright © 2008 John Wiley & Sons, Ltd.     

Aperture based Raman spectroscopy on SiGe film structures with high spatial resolution

We have investigated silicon–germanium (SiGe) line structures employing metallic apertures in combination with Raman spectroscopy to obtain high‐spatial strain resolution below the diffraction limit. The apertures were cut into specifically shaped electrochemically etched tungsten tips, which were adjusted within the Raman laser beam on the sample surface by a tuning fork atomic force microscope. With this setup, line structures on patterned SiGe films with a center‐to‐center distance down to 200 nm were resolved in the Raman scans, evidently indicating a resolution clearly below the far‐field Raman resolution of about 600 nm for the used instrument. This setup allows improved local strain analysis by Raman spectroscopy and shows potential for further near‐field Raman applications. Copyright © 2008 John Wiley & Sons, Ltd.     

Multivariate analysis applied to particle‐induced X‐ray emission mapping

The use of particle‐induced X‐ray emission (PIXE) for elemental speciation and quantification has gained new attention thanks to mapping capabilities. Microprobes are able to raster a proton beam and produce elemental maps on the micrometre scale. Moreover, recent developments of in‐air PIXE instrumentation have enabled the acquisition of large area elemental maps. However, the amount of data produced is very large, and the data processing is not trivial. In this paper, we propose the use of multivariate analysis to process data of PIXE mapping. First, we apply the non‐negative matrix factorization (NMF), which is a nonsupervised machine‐learning algorithm, to decompose the data into a smaller number of components; then, we use the k‐means algorithm to divide the pixels into categories regarding similarities in the NMF results; finally, we sum the spectra of all pixels in the same category so that the results can be analyzed by standard procedures for PIXE quantification. This last step is important to enable the quantification of the elements found in each component by correctly accounting for matrix self‐absorptions. With the procedure described in this paper, not just, we reduced the number of variables, facilitating the reasoning process on the data by employing the multivariate analysis, but we also increased the counting statistics by summing similar pixels leading to better results concerning the quantification of trace elements. We also propose methods for both, the automatic determination of the optimal number of components to describe the dataset, and for the combined analysis of multiple detectors.     

A new microfocus x‐ray source,iMOXS, for highly sensitive XRF analysis in scanning electron microscopes

Scanning electron microscopes are usually equipped with energy‐dispersive X‐ray detectors for electron probe microanalysis. This widespread analytical method allows investigators to determine the elemental composition of specimens with a spatial resolution of about 1 µm. However, owing to the electron–specimen interaction, the emitted spectra reveal, in addition to characteristic lines, also a high level of continuous bremsstrahlung background. As a result, elements with low concentrations cannot be identified. The minimum detection limit can be diminished by two orders of magnitude if the characteristic lines are excited as fluorescence by an additional x‐ray source. In this case, the emergence of bremsstrahlung is considerably reduced. Combining a high‐brilliance microfocus x‐ray tube with efficient polycapillary optics enables one to realize an experimental arrangement for performing local fluorescence analysis at the same point where the electron beam hits the sample. The polycapillary optics under consideration focuses the emitted x‐radiation onto focal spots between 30 and 100 µm in diameter. Count rates of several thousands cps have been achieved. Elemental maps have been obtained by means of the motorized specimen stage of the microscope. Copyright © 2005 John Wiley & Sons, Ltd.     

A device for X‐Ray elemental mapping using annular radioisotope source

An energy‐dispersive system is described for elemental mapping by X‐ray fluorescence spectrometry. The present study describes the design of an X‐ray fluorescence spectrometer and presents its performance in elemental mapping applications. The spectrometer is based on a new ring‐shaped collimator with a pinhole in the center of it and a ring‐shaped Am‐241 isotope mounted in the collimator as a source for excitation of X‐ray fluorescence. The photons were detected by high‐resolution Si (Li) detector coupled to a multi‐channel analyser and cooled by liquid nitrogen. In this study, we used two samples; one of them was made from pure elemental powders, and the second one was a piece of a stone and three types of maps were plotted. In the maps type one, the areas of the elements were shown with a single color. These maps only show the location of the elements in the sample. In the maps type two, the area of each element was shown with different colors because of the count (intensity) related to the area. In the third type of the maps for each element, depending on the elements' position on the sample, the counts were plotted in three dimensions. The areas with higher intensity have greater height, and areas with lower intensity have lower altitude. These two last types of maps provide information about the homogeneity or heterogeneity of the elemental distribution in the samples. The spectrometer can perform non‐destructive analyses of samples and objects in the air. Copyright © 2017 John Wiley & Sons, Ltd.     

New directions in X-ray microscopy

The development of high brightness X-ray sources and high resolution X-ray optics has led to rapid advances in X-ray microscopy. Scanning microscopes and full-field instruments are in operation at synchrotron light sources worldwide, and provide spatial resolution routinely in the 25–50 nm range using zone plate focusing elements. X-ray microscopes can provide elemental maps and/or chemical sensitivity in samples that are too thick for electron microscopy. Lensless techniques, such as diffraction microscopy, holography and ptychography are also being developed. In high resolution imaging of radiation-sensitive material the effects of radiation damage needs to be carefully considered. This article is designed to provide an introduction to the current state and future prospects of X-ray microscopy for the non-expert.     

Total reflection x‐ray fluorescence analysis—a review

Total reflection x‐ray fluorescence analysis (TXRF) is a special energy‐dispersive x‐ray analytical technique extending XRF down to the ultra trace element range. Detection limits of picograms or nanograms per gram levels are reached with x‐ray tube excitation. Using synchrotron radiation as excitation source, femtogram levels are detectable, particularly important for Si wafer surface analysis. TXRF is specially suited for applications in which only a very small amount of sample is available, as only a few micrograms are required for the analysis. In this review, an overview of theoretical principles, advantages, instrumentation, quantification and application is given. Chemical analysis as well as surface analysis including depth profiling and thin‐film characterization is described. Special research results on extension to low‐Z elements, excitation with synchrotron radiation and x‐ray absorption spectroscopy (XAS) for chemical speciation at trace levels are reviewed. Copyright © 2007 John Wiley & Sons, Ltd.     

X‐ray spectromicroscopy in soil and environmental sciences

X‐ray microscopy is capable of imaging particles in the nanometer size range directly with sub‐micrometer spatial resolution and can be combined with high spectral resolution for spectromicroscopy studies. Two types of microscopes are common in X‐ray microscopy: the transmission X‐ray microscope and the scanning transmission X‐ray microscope; their set‐ups are explained in this paper. While the former takes high‐resolution images from an object with exposure times of seconds or faster, the latter is very well suited as an analytical instrument for spectromicroscopy. The morphology of clusters or particles from soil and sediment samples has been visualized using a transmission X‐ray microscope. Images are shown from a cryo‐tomography experiment based on X‐ray microscopy images to obtain information about the three‐dimensional structure of clusters of humic substances. The analysis of a stack of images taken with a scanning transmission X‐ray microscope to combine morphology and chemistry within a soil sample is shown. X‐ray fluorescence is a method ideally applicable to the study of elemental distributions and binding states of elements even on a trace level using X‐ray energies above 1 keV.     

Confocal Raman spectroscopy to monitor intracellular penetration of TiO2 nanoparticles

Confocal Raman microscopy, a noninvasive, label‐free, and high‐spatial resolution imaging technique, in combination with K‐mean cluster analysis and a correlation coefficient map, was employed to trace titanium dioxide (TiO₂) nanoparticles in living MCF‐7 and TERT cells. The penetration of TiO₂ nanoparticles into cells revealed a gradual time‐dependent diffusion of nanoparticles over the entire cell. Cell apoptosis was monitored by tracing cytochrome c diffusion into the cytoplasm. A comparison with the mitochondrial clustering indicated that cytochrome c was inside the mitochondria for TiO₂ concentration of 2 µg ml⁻¹. This result demonstrates that the presence of TiO₂ particles within a cell does not induce apoptosis. We demonstrated that confocal Raman microscopy allow to follow penetration of TiO₂ particles in cell and to monitor the apoptotic status of the penetrated cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Evaluation of the improved three‐dimensional resolution of a synchrotron radiation computed tomograph using a micro‐fabricated test pattern

A micro test pattern prepared by focused ion beam milling was used to evaluate the three‐dimensional resolution of a microtomograph at the BL20B2 beamline of SPring‐8. The resolutions along the direction within the tomographic slice plane and perpendicular to it were determined from the modulation transfer functions. The through‐plane resolution perpendicular to the tomographic slice was evaluated to be 8 µm, which corresponds to the spatial resolution of two‐dimensional radiographs. In contrast, the in‐plane resolution within the slice was evaluated to be 12 µm. Real‐space interpolation was performed prior to the tomographic reconstruction, giving an improved in‐plane resolution of 8.5 µm. However, the 8 µm pitch pattern was resolved in the interpolated slice image. To reflect this result, another resolution measure from the peak‐to‐valley difference plot was introduced. This resolution measure gave resolution limits of 7.4 µm for the in‐plane direction and 6.1 µm for the through‐plane direction. The three‐dimensional test pattern along with the interpolated reconstruction enables the quantitative evaluation of the spatial resolution of microtomographs.     

Broad‐beam PIXE and µ‐PIXE analysis of normal and in vitro demineralized dental enamel

Dental enamel has been widely studied by particle‐induced x‐ray emission (PIXE), but less attention was paid to its demineralization, which leads to caries formation. Using broad‐beam PIXE and µ‐PIXE, we investigated normal enamel and the in vitro formation of pre‐carious lesion in lactic acid solution, aiming also to evaluate intercusp differences within the same tooth. Broad‐beam PIXE was performed using 3.0 MeV protons, and µ‐PIXE maps of Ca, Fe and Zn were collected with 3.1 MeV protons at ～4 µm resolution. In normal enamel a differentiated Ca‐rich surface layer was observed, where Fe and Zn reached their highest levels. In deeper layers, Fe and Zn evidenced quasiperiodic patterns of maxima, possibly due to coupled diffusion‐reaction catalytic processes involved in the enamel growth. Both Fe and Zn appeared to be located in a few distinct types of pools. Near the surface, demineralization induced an increase of Fe, Cu, Zn, Sr and Pb with respect to Ca, attributed to partial hydroxyapatite dissolution and/or to chelate extraction and concentration of trace metals. Ca maps revealed limited changes in the surface layer and a massive loss in the inner enamel; here, Fe was almost depleted and Zn partially removed. The maps of Ca, Fe and Zn demonstrated major intercusp variations in both normal and altered enamel. Thus, broad‐beam PIXE and µ‐PIXE, which do not require (semi)thin sectioning of the tooth as the conventional methods, provide compositional and structural insight of normal dental enamel, of its intercusp variability and of the alterations produced by in vitro demineralization, largely not accessible to the current techniques, and highly relevant for understanding the incipient caries formation. Copyright © 2008 John Wiley & Sons, Ltd.     

Fast XANES fluorescence imaging using a Maia detector

A new fast X‐ray absorption spectroscopy scanning method was recently implemented at the Hard X‐ray Microprobe endstation P06, PETRA III, DESY, utilizing a Maia detector. Spectromicroscopy maps were acquired with spectra for X‐ray absorption near‐edge structure (XANES) acquisition in the sub‐second regime. The method combines XANES measurements with raster‐scanning of the sample through the focused beam. The order of the scanning sequence of the axes, one beam energy axis and two (or more) spatial axes, is a variable experimental parameter and, depending on it, the dwell at each location can be either single and continuous (if the energy axis is the inner loop) or in shorter discontinuous intervals (if a spatial axis is innermost). The combination of improved spatial and temporal resolution may be necessary for rapidly changing samples, e.g. for following in operando chemical reactions or samples highly susceptible to beam damage where the rapid collection of single XANES spectra avoids issues with the emergence of chemical changes developing from latent damage. This paper compares data sets collected on a specially designed test pattern and a geological thin‐section scanning the energy as inner, middle and outer axis in the sequence. The XANES data of all three scanning schemes is found to show excellent agreement down to the single‐pixel level.     

Synchrotron-based X-ray fluorescence,imaging and elemental mapping from biological samples

The present study utilized the new hard X-ray microspectroscopy beamline facility, X27A, available at NSLS, BNL, USA, for elemental mapping. This facility provided the primary beam in a small spot of the order of ∼10 μm, for focussing. With this spatial resolution and high flux throughput, the synchrotron-based X-ray fluorescent intensities for Mn, Fe, Zn, Cr, Ti and Cu were measured using a liquid-nitrogen-cooled 13-element energy-dispersive high-purity germanium detector. The sample is scanned in a ‘step-and-repeat’ mode for fast elemental mapping measurements and generated elemental maps at 8, 10 and 12 keV, from a small animal shell (snail). The accumulated trace elements, from these biological samples, in small areas have been identified. Analysis of the small areas will be better suited to establish the physiology of metals in specific structures like small animal shell and the distribution of other elements.     

Experimental evaluation of X‐ray optics applied for microanalysis

The performance of x‐ray capillary lenses has been evaluated. The tests were carried out using an x‐ray tube set‐up. A single glass capillary with tapered inner channel, a monolithic glass polycapillary, and an in‐house manufactured single metallic capillary with parabolic inner channel were characterized in terms of gain, spatial resolution, and element detection limits. The spatial resolution of a confocal set‐up utilizing a monolithic glass polycapillary and a polycapillary conical collimator has also been measured. The highest gain of about 2500 was observed for the glass polycapillary. The maximum gain achieved with the single glass capillary was equal to about 25, and the gain of the metallic capillary was only slightly greater than 1. For the glass capillary and polycapillary lenses, significant filtering of the higher‐energy photons (energy > 8 keV) was observed. The lowest relative detection limits were obtained with an ordinary cylindrical collimator and the polycapillary lens. Similar absolute detection limits were achieved with the use of the polycapillary and single capillary lenses. A relation between the ratios of the detection limits of elements achieved with different x‐ray lenses and the lens parameters (spatial resolution and gain) has been proposed and was verified experimentally. The monolithic polycapillary lens was found to be an optimum focusing device for an x‐ray tube‐based scanning spectrometer. This type of x‐ray lens can be coupled with a polycapillary conical collimator or a polycapillary half‐lens to make a confocal x‐ray microscope capable of depth profiling with a spatial resolution equal to about 30 micrometers. Copyright © 2008 John Wiley & Sons, Ltd.     

Increased zinc accumulation in mineralized osteosarcoma tissue measured by confocal synchrotron radiation micro X‐ray fluorescence analysis

Abnormal tissue levels of certain trace elements such as zinc (Zn) were reported in various types of cancer. Little is known about the role of Zn in osteosarcoma. Using confocal synchrotron radiation micro X‐ray fluorescence analysis, we characterized the spatial distribution of Zn in high‐grade sclerosing osteosarcoma of nine patients (four women/five men; seven knee/one humerus/one femur) following chemotherapy and wide surgical resection. Levels were compared with adjacent normal tissue. Quantitative backscattered electron imaging as well as histological examinations was also performed. On average, the ratio of medians of Zn count rates (normalized to calcium) in mineralized tumor tissue was about six times higher than in normal tissue. There was no difference in Zn levels between tumor fraction areas with a low fraction and a high fraction of mineralized tissue, which were clearly depicted using quantitative backscattered electron imaging. Moreover, we found no correlation between the Zn values and the type of tumor regression according to the Salzer‐Kuntschik grading. The underlying mechanism of Zn accumulation remains unclear. Given the emerging data on the role of trace elements in other types of cancer, our novel results warrant further studies on the role of trace elements in bone cancer. Copyright © 2016 The Authors. X‐Ray Spectrometry published by John Wiley & Sons Ltd.     

Elemental distribution by synchrotron X-ray microfluorescence of prostate 3D cell culture

Prostate cancer is a highly prevalent disease and ranks second among malignant neoplasms that affect men around the world, behind lung cancer alone. Trace elements are very important and are involved in many cellular processes. The X-ray microfluorescence technique is an advanced tool of high spatial resolution, sensitivity, multielemental analysis, and nondestructiveness for trace element study. This study aimed to investigate the elemental distribution in spheroids obtained through the following human prostate cell lines using synchrotron X-ray microfluorescence: tumor cell line androgen independent (DU145), tumor cell line androgen dependent (LNCaP), and normal cell line (RWPE-1). The measurements were performed with a standard geometry of 45° of incidence, excited by a white beam using a pixel of 25 μm and an acquisition time of 300 ms/pixel at the X-ray fluorescence beamline at the Synchrotron Light National Laboratory (Campinas, Brazil). The synchrotron X-ray microfluorescence results showed differences between groups in all elements analyzed and suggested that further studies should be performed to understand the relationship of these trace elements with the progression and development of the disease.     

Ultraviolet tip‐enhanced nanoscale Raman imaging

We have constructed an ultraviolet (UV)‐apertureless near‐field scanning optical microscope‐Raman spectroscopy system by using an aluminum tip for the simultaneous measurement of topography and Raman scattering of nanomaterials with high spatial resolution. The topography, Rayleigh scattering image, and tip‐enhanced Raman scattering image of the carbon nanotube film showed that a spatial resolution of around 19 nm was achieved. This spatial resolution of UV‐Raman mapping image exceeds that of previous approaches, which have several hundred nanometers of spatial resolution. Copyright © 2012 John Wiley & Sons, Ltd.     

Synchrotron fluorescence nanoimaging of a single Co‐implanted ZnO nanowire

In this study, we report the application of synchrotron radiation nanoprobe technique to the elemental analysis of single as‐grown and Co‐implanted ZnO nanowires. The nano‐X‐ray fluorescence technique enabled us not only to examine the spatial variation of Zn and Co elements, but also to disregard the presence of residual impurities in the nanowires, as well as the detection of Fe and Sn residual impurities in the substrates. Our observations provide strong evidence for the overall elemental uniformity of Zn and Co along the wires, without clustering or segregation effects. Within the nanoprobe spatial resolution, our findings indicate a Co localization within thicker irregularities observed with scanning electron microscopy. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantitative magnetic resonance flow and diffusion imaging in porous media

Quantitative flow and diffusion measurements have been made for water in model porous media, using magnetic resonance micro-imaging methods. The samples consisted of compacted glass beads of various sizes down to 1 mm diameter. Typical flow and diffusion images exhibited a spatial resolution of 117 μm × 117 μm and velocities in the range 1–2 mm/s. Comparison of volume flow rates calculated from the flow velocity maps with values measured directly yielded good agreement in all cases. There was also good agreement between the mean diffusion coefficient of water calculated from the diffusion maps and the bulk diffusion coefficient for pure water at the same temperature. In addition, the mean diffusion coefficient did not depend on the pore sizes in the bead diameter range of 1–3 mm. Our results also show that partial volume effects can be compensated by appropriate thresholding of the images prior to the final Fourier transformation in the flow-encoding dimension.     

A sub‐micrometer resolution hard X‐ray microprobe system of BL8C at Pohang Light Source

A microprobe system has been installed on the nanoprobe/XAFS beamline (BL8C) at PLS‐II, South Korea. Owing to the reproducible switch of the gap of the in‐vacuum undulator (IVU), the intense and brilliant hard X‐ray beam of an IVU can be used in X‐ray fluorescence (XRF) and X‐ray absorption fine‐structure (XAFS) experiments. For high‐spatial‐resolution microprobe experiments a Kirkpatrick–Baez mirror system has been used to focus the millimeter‐sized X‐ray beam to a micrometer‐sized beam. The performance of this system was examined by a combination of micro‐XRF imaging and micro‐XAFS of a beetle wing. These results indicate that the microprobe system of the BL8C can be used to obtain the distributions of trace elements and chemical and structural information of complex materials.