Elemental mapping of frozen‐hydrated cells with cryo‐scanning X‐ray fluorescence microscopy

Visualizing the elemental distributions of cells and tissues is of growing importance in biology and medical science because such data deepen our understanding of the behavior of metal‐binding proteins and ions. Elemental mapping by X‐ray fluorescence analysis with a hard X‐ray nanobeam is very well suited for this purpose owing to its high sensitivity and high resolution. Using this technique, samples must be prepared without artifacts that are caused by treatments such as chemical fixation and staining procedures. In many studies of elemental mapping, sample preparation is not explicitly considered. To overcome this deficiency, we developed a cryo‐scanning X‐ray fluorescence microscope and installed it in the second experimental hutch of BL29XUL of SPring‐8. We used it to observe frozen‐hydrated cells that had been fixed by a quick‐freezing technique to preserve elemental data of the living state at an X‐ray energy of 11.5 keV. The distributions of K, Ca, Fe, Cu and Zn were successfully visualized. The distributions of these elements (especially those of K, Ca and Fe) differed from those in cells fixed with paraformaldehyde. Copyright © 2010 John Wiley & Sons, Ltd.     

Methodological challenges of optical tweezers‐based X‐ray fluorescence imaging of biological model organisms at synchrotron facilities

Recently, a radically new synchrotron radiation‐based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non‐contact laser‐based sample manipulation with synchrotron radiation confocal X‐ray fluorescence (XRF) microimaging for the first time at ESRF‐ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF‐related challenges are reported. In general, the applicability of the OT‐based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi‐elemental analysis is of relevance at the (sub)micrometre spatial resolution level.     

Large‐surface‐area diamond (111) crystal plates for applications in high‐heat‐load wavefront‐preserving X‐ray crystal optics

Fabrication and results of high‐resolution X‐ray topography characterization of diamond single‐crystal plates with large surface area (10 mm × 10 mm) and (111) crystal surface orientation for applications in high‐heat‐load X‐ray crystal optics are reported. The plates were fabricated by laser‐cutting of the (111) facets of diamond crystals grown using high‐pressure high‐temperature methods. The intrinsic crystal quality of a selected 3 mm × 7 mm crystal region of one of the studied samples was found to be suitable for applications in wavefront‐preserving high‐heat‐load crystal optics. Wavefront characterization was performed using sequential X‐ray diffraction topography in the pseudo plane wave configuration and data analysis using rocking‐curve topography. The variations of the rocking‐curve width and peak position measured with a spatial resolution of 13 µm × 13 µm over the selected region were found to be less than 1 µrad.     

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.     

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.     

Depth‐selective elemental imaging of microSD card by confocal micro XRF analysis

A semiconductor device, a microSD card, was measured by using two XRF instruments. 2D elemental images were obtained using a micro‐XRF system with a spatial resolution of 10 µm. Elemental distributions of the near‐surface region of the sample were clearly shown. Titanium was observed in the resin constituting the sample. Nickel and gold were observed on a terminal and localization of the sample. Elemental distribution of copper reflected the circuit structure of the measurement area that was in the neighborhood of the sample surface. Moreover, the elemental depth distributions of the sample were measured by using a confocal micro‐XRF instrument. The confocal micro‐XRF instrument was constructed in the laboratory with fine‐focus polycapillary x‐ray optics. The depth resolution of the developed spectrometer was 13.7 µm at an energy of Au Lβ (11.4 keV). The elemental images obtained at near‐surface by confocal micro‐XRF were the same as the results obtained from 2D micro‐XRF. However, different Cu images were obtained at a depth of several tens of micrometers. This indicates that microSD cards consist of a few different Cu‐circuit structure designs. The elemental depth distributions of each circuit structure of the semiconductor device were clearly shown by confocal micro‐XRF. Copyright © 2013 John Wiley & Sons, Ltd.     

Characteristics of a robust and portable large area X‐ray fluorescence imaging system

A low‐cost and portable Energy Disperse X‐Ray Fluorescence imaging system is presented. It is capable of scanning large areas (up to 10 × 10 cm²) in steps as small as 0.2 mm. It consists of a small X‐ray tube and a Silicon Drift Detector, both controlled by a homemade software. The spatial resolution is evaluated as a function of the step width and of the X‐ray collimator diameter. The time of acquisition per unit of scanned surface is studied as a function of the step width and of the time of acquisition per point. X‐ray fluorescence images showing the 2D distributions of some elements in biological samples are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

Kβ/Kα x‐ray intensity ratios for bromine and iodine compounds

Kβ/Kα x‐ray intensity ratios of some Br and I compounds were studied. The samples were excited by 59.5 keV γ‐rays emitted from an Am‐241 radioisotope source and characteristic K x‐rays emitted from the samples were counted by means of an Si(Li) detector which has a resolution 155 eV at 5.9 keV. The experimental values were compared with the calculated theoretical values for elemental Br and I. Copyright © 2003 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)     

Integrating 3D images using laboratory‐based micro X‐ray computed tomography and confocal X‐ray fluorescence techniques

The application of non‐destructive imaging to characterizing samples has become more important as the costs of samples increase. Imaging a sample via X‐ray techniques is preferable when altering or even touching the sample affects its properties, or when the sample is fielded after characterization. Two laboratory‐based X‐ray techniques used at Los Alamos include micro X‐ray computed tomography (MXCT) and confocal micro X‐ray fluorescence (confocal MXRF). Both methods create a 3D rendering of the sample non‐destructively. MXCT produces a high‐resolution (sub‐µm voxel) rendering of the sample based upon X‐ray absorption; the resulting model is a function of density and does not contain any elemental information. Confocal MXRF produces an elementally specific 3D rendering of the sample, but at a lower (30 × 30 × 65 µm) resolution. By combining data from these two techniques, scientists provided a more comprehensive method of analysis. We will describe a MATLAB routine written to render each of these data sets individually and/or within the same coordinate system. This approach is shown in the analysis of two samples: an integrated circuit surface mounted resistor and a machined piece of polystyrene foam. The samples chosen provide an opportunity to compare and contrast the two X‐ray techniques, identify their weaknesses and show how they are used in a complementary fashion. Copyright © 2010 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.     

Nondestructive elemental depth profiling of Japanese lacquerware ‘Tamamushi‐nuri’ by confocal 3D‐XRF analysis in comparison with micro GE‐XRF

We have applied recently two XRF (micro x‐ray fluorescence) methods [micro‐Grazing Exit XRF (GE‐XRF) and confocal 3D‐XRF] to Japanese lacquerware ‘Tamamushi‐nuri.’ A laboratory grazing‐exit XRF (GE‐XRF) instrument was developed in combination with a micro‐XRF setup. A micro x‐ray beam was produced by a single capillary and a pinhole aperture. Elemental x‐ray images (2D images) obtained at different analyzing depths by micro GE‐XRF have been reported. However, it was difficult to directly obtain depth‐selective x‐ray spectra and 2D images. A 3D XRF instrument using two independent polycapillary x‐ray lenses and two x‐ray sources (Cr and Mo targets) was also applied to the same sample. 2D XRF images of a Japanese lacquerware showed specific distributions of elements at the different depths, indicating that ‘Tamamushi‐nuri’ lacquerware has a layered structure. The merits and disadvantages of both the micro GE‐XRF and confocal micro XRF methods are discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

A scanning transmission X‐ray microscope at the Pohang Light Source

A scanning transmission X‐ray microscope is operational at the 10A beamline at the Pohang Light Source. The 10A beamline provides soft X‐rays in the photon energy range 100–2000 eV using an elliptically polarized undulator. The practically usable photon energy range of the scanning transmission X‐ray microscopy (STXM) setup is from ～150 to ～1600 eV. With a zone plate of 25 nm outermost zone width, the diffraction‐limited space resolution, ～30 nm, is achieved in the photon energy range up to ～850 eV. In transmission mode for thin samples, STXM provides the element, chemical state and magnetic moment specific distributions, based on absorption spectroscopy. A soft X‐ray fluorescence measurement setup has been implemented in order to provide the elemental distribution of thicker samples as well as chemical state information with a space resolution of ～50 nm. A ptychography setup has been implemented in order to improve the space resolution down to 10 nm. Hardware setups and application activities of the STXM are presented.     

A gas proportional‐scintillation counter for x‐ray spectrometry in the 0.1–3 keV range

Results for the energy resolution of a uniform‐field gas proportional‐scintillation counter are presented for the range 0.1–3 keV. A comparison with results from the literature shows that the detector is most suitable for this energy range. Examples of x‐ray fluorescence pulse‐height distributions from geological and industrial samples are presented. Copyright © 2004 John Wiley & Sons, Ltd.     

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.     

In vitro evaluation of osteoblast‐like cells on hydroxyapatite‐coated porous stainless steel implants by synchrotron radiation x‐ray fluorescence

The successful application of artificial implants requires osseointegration in the implanted structures. To stimulate bone growth, synthetic hydroxyapatite obtained by the coprecipitation process was coated onto porous stainless steel substrates in order to enhance the biocompatibility and, consequently, mineralization. The substrate of choice was porous 316L stainless steel for its high resistance, mechanical strength and low density due to its foam structure. The aim of the present study was to investigate the biological response of the fabricated implants cultured with MC3T3‐E1 mouse osteoblast‐like cells by analyzing the variation in the elemental concentration, mainly calcium, along with the cellular differentiation and mineralization. By employing synchrotron radiation x‐ray fluorescence spectroscopy (SRXRF), intracellular elemental distribution and concentration could be determined, revealing a clear increase in the total calcium content. This preliminary data suggests that synthetic hydroxyapatite on porous stainless steel substrates might be successfully used for biocompatible medical implants. Copyright © 2009 John Wiley & Sons, Ltd.     

Measurements of Coster–Kronig enhancement factors of some elements in the atomic number range 66 ≤ Z ≤ 72

The Lι, Lα, Lβ and Lγ x‐ray production cross‐sections in four elements with Z ranging from 66 to 72 at seven different energies in the interval 8.265–11.730 keV were measured. Experimental measurements were carried out on a few elemental samples to examine the effect of Coster–Kronig transitions on fluorescence cross‐sections for the L x‐ray line using an Si(Li) detector system with an energy resolution of 160 eV at 5.96 keV x‐ray energy. Absolute values of L x‐ray cross‐sections were calculated with incorporation of the enhancement due to the Coster–Kronig effect for these elements. The measured enhancement factors are smaller than predicted by theory. Copyright © 2003 John Wiley & Sons, Ltd.     

Improvement of spatial resolution of µ‐XRF by using a thin metal filter

An improvement of spatial resolution of µ‐XRF by using a thin metal filter was investigated. The size of the x‐ray beam focused by the polycapillary x‐ray lens depended on the energy of the characteristic x‐rays. Original spot sizes at the focal point were 48 µm for CrKα, 41 µm for NiKα, and 28 µm for MoKα, respectively. To make the x‐ray beam size small, Ti? Cu thin foil was placed between the output of the lens and the focal point as a metal filter to reduce the continuous x‐rays. Finally, the x‐ray microbeam size was improved to 30 µm by applying a filter. Clear 2D mapping images of Cr, Fe, and Ni in 300‐mesh stainless steel could be obtained by applying this filter. Copyright © 2008 John Wiley & Sons, Ltd.     

Enhancements in full‐field PIXE imaging—Large area elemental mapping with increased lateral resolution devoid of optics artefacts

The combination of a pn‐junction charge‐coupled device‐based pixel detector with a poly‐capillary X‐ray optics was installed and examined at the Helmholtz‐Zentrum Dresden‐Rossendorf. The set‐up is intended for particle‐induced X‐ray emission imaging to survey the trace elemental composition of flat/polished geological samples. In the standard configuration, a straight X‐ray optics (20 μm capillary diameter) is used to guide the emitted photons from the sample towards the detector with nearly 70 000 pixels. Their dimensions of 48 × 48 μm² are the main limitation of the lateral resolution. This limitation can be bypassed by applying a dedicated subpixel algorithm to recalculate the footprint of the photon's electron cloud in the detector. The lateral resolution is then mainly determined by the capillary's diameter. Nevertheless, images are still superimposed by the X‐ray optics pattern. The optics' capillaries are grouped in hexagonal bundles resulting in a reduced transmission of X‐rays in the boundary regions. This influence can be largely suppressed by combining a series of short measurements at slightly shifted positions using a precision stage and correcting the image data for this shifting. The use of a subpixel grid for the image reconstruction allows a further increase of the spatial resolution. This approach of image‐stacking and multiframe super‐resolution in combination with the subpixel correction algorithm is presented and illustrated with experimental data. Additionally, a flat‐field correction is shown to remove the remaining imaging inhomogeneity caused by non‐uniform X‐ray transmission. The described techniques can be used for all X‐ray spectrometry methods using an X‐ray camera to obtain high‐quality elemental images.