Integrated Three-Dimensional Microanalysis Combining X-Ray

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Integrated three dimensional micro-analysis combining Xray microtomography and X-ray fluorescence methodologies Brecht Laforce, Bert Masschaele, Matthieu N. Boone, David Schaubroeck, Manuel Dierick, Bart Vekemans, Christophe Walgraeve, Colin R. Janssen, Veerle Cnudde, Luc Van Hoorebeke, and Laszlo Vincze Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03205 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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Analytical Chemistry

Integrated three dimensional micro-analysis combining X-ray microtomography and X-ray fluorescence methodologies Brecht LAFORCE1,*, Bert MASSCHAELE2,3, Matthieu N. BOONE2, David SCHAUBROECK4, Manuel DIERICK2, Bart VEKEMANS1, Christophe WALGRAEVE5, Colin JANSSEN6, Veerle CNUDDE7, Luc VAN HOOREBEKE2, Laszlo VINCZE1 1. X-ray Microspectroscopy and Imaging group (XMI), Department of Analytical Chemistry, Ghent University, Krijgslaan 281 S12, b9000 Ghent, Belgium 2. UGCT-Department of Physics and Astronomy, Ghent University, Proeftuinstraat 86, B-9000 Ghent, Belgium 3. X-Ray Engineering (XRE) bvba, Technologiepark 5, B-9052 Zwijnaarde, Belgium

4. Center for Microsystems Technology (CMST), imec and Ghent University, Technologiepark 15, 9052 Ghent, Belgium 5. Department of sustainable organic chemistry and technology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium 6. Laboratory of Environmental Toxicology and Aquatic Ecology , Ghent University, Coupure links 653, 22, B-9000 Ghent, Belgium 7. UGCT-PProGRess, Department of geology, Ghent University, Krijgslaan 281 S8, B-9000 Ghent

ABSTRACT: A novel 3D elemental and morphological analysis approach is presented combining X-ray µCT, XRF tomography and confocal XRF analysis in a single laboratory instrument (Herakles). Each end station of Herakles (µCT, XRF-CT and confocal XRF) represents the state-of-the-art of currently available laboratory techniques. The integration of these techniques enables linking the (quantitative) spatial distribution of chemical elements within the materials investigated to their three-dimensional (3D) internal morphology/structure down to 1-10 µm resolution level, which has not been achieved so-far using laboratory X-ray techniques. The concept of Herakles relies strongly on its high precision (around 100 nm) air-bearing motor system that connects the different end-stations, allowing combined measurements based on the above X-ray techniques while retaining the coordinate system. Inhouse developed control and analysis software further ensures a smooth integration of the techniques. Case studies on a Cu test pattern, a Daphnia magna model organism and a perlite biocatalyst support material demonstrate the attainable resolution, elemental sensitivity of the instrument and the strength of combining these three complementary methodologies.

In various scientific disciplines (e.g. cosmo- and geochemistry, environmental science, cultural heritage), there is a strong demand to develop techniques that enable linking the (quantitative) spatial distribution of chemical elements within the materials investigated to their three-dimensional (3D) internal morphology/structure down to the (sub-)micrometer resolution level. The integrated approach in a laboratory setting has not been demonstrated so far, although it has the potential to provide a highly detailed 3D view of the structural and compositional properties of the investigated materials with a spatial resolution reaching the micron scale and below in a nondestructive manner. Since the discovery of X-rays by W.C. Röntgen in 1895, this type of radiation is at the heart of numerous nondestructive imaging and chemical analysis techniques. Immediately after the discovery, X-rays were used for radiographic imaging, revealing a 2D projection of the interior of an object. X-ray imaging was extended to 3D in the late 1970s by the discovery of Computed Tomography (CT), which soon became a very popular medical diagnostics tool and swiftly found its way into academic and industrial applications. For these applications, the advent of high-resolution X-ray tomography (µCT) can be considered a breakthrough.1-5 This 3D

imaging technique is now becoming a well-known analysis methodology in numerous applications, reaching resolutions below 1 µm in laboratory-based setups and well below 100 nm at synchrotron radiation facilities.6-8 Besides imaging, X-rays were early on deployed for chemical and physical material characterization as well. In the 1910s, X-rays were first used to determine the crystallographic structure of materials based on X-ray diffraction (XRD). At the end of the 1920s X-rays were proposed as primary beam to excite fluorescent radiation for elemental analysis based on Moseley’s law, which links the X-ray energies of the fluorescent lines to the atomic number of the corresponding elements. Both XRD and XRF have since evolved beyond recognition, and are used in life sciences, material sciences, archaeology, … Classically, X-ray fluorescence (XRF) was a bulk analysis technique, later evolving to a 2D elemental imaging methodology by scanning the sample with a small, focused X-ray beam. However, during the last decades three-dimensional XRF methodologies have been developed, either by using specialized optics on both the X-ray source and detector (confocal XRF analysis)9,10 or based on pencil-shaped primary X-ray beams and reconstruction algorithms similar to the ones used in µCT (i.e. XRF-CT).11-19 These 3D XRF techniques originated at synchrotron radiation

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facilities, which are currently at the forefront of new developments in X-ray based methodologies because of their superior X-ray beam properties as compared to laboratory instrumentation (e.g. high collimation and intensity). Recent developments on laboratory X-ray sources, efficient focusing optics and detectors have made 3D XRF also feasible at the laboratoryscale. Although these X-ray based techniques are very powerful, they are limited to their respective outcome, i.e. 3D morphology for transmission-based CT, chemical composition for X-ray fluorescence and crystal structure for X-ray crystallography. To overcome this limitation, recent developments in hardware and software are proceeding toward a combined use of these techniques and the development of hybrid techniques. At large-scale synchrotron radiation facilities, the coherence of the X-ray beam, both spatial and spectral, is particularly advantageous for the development of such methodologies. In recent years, techniques such as µCT combined with confocal XRF,20 3D-X-ray diffraction ,21-23 coherent diffractive imaging 24,25 and XRF-CT11,26,27 have been developed, crossing the borders between the three methodologies described above. Most of these examples use synchrotron radiation based techniques. Access to these synchrotron facilities is scarce and operational costs are high. Researchers have therefore investigated possibilities to bring these combined techniques to the lab. This has been successfully implemented for e.g. hyperspectral imaging28,29, XRF-CT12, 3D-XRD21,30 and a combined system for µCT and full-field XRF26,27. Some of these methods are still under development or exhibit extreme limitations in terms of sample variation. Furthermore, in many cases the combined data is merely fused, and mutual feedback is limited. In this paper, we present a unique laboratory-based system, the Herakles 3D X-ray scanner, which combines highresolution X-ray transmission tomography (µCT), confocal Xray fluorescence (cXRF) and X-ray fluorescence tomography (XRF-CT), developed and built at Ghent University. The integration of the three different imaging end-stations is realized through a very high precision motor stage in order to actively couple the data from the different techniques, allowing for optimal use of the measured data. The potential of the instrument is demonstrated using three case studies: a Cu on silica test structure (comparable to a microchip), the Daphnia magna model organism and a perlite biocatalyst support material.

Materials and Methods High-resolution X-Ray Computed Tomography The µCT setup (Figure 1) is equipped with an open transmission-type microfocus X-ray tube (X-RAY WorX, Garbsen, Germany) with a high resolution tungsten target. Voltages between 20 and 100 kV can be applied, with a maximum target power of 10 W when using a microfocus spot. The smallest achievable spot size is 700 nm, as specified by the manufacturer, the voxel size during typical scans on real-life samples is around 1 µm. The tube head is water cooled to reduce thermal dimension changes during a µCT scan. The setup is furnished with two different X-ray detectors. For the imaging of low-attenuating samples, a Photonic Science VHR CCD sensor (Photonic Science, Millham, UK) is used. This cooled CCD detector has 4008 x 2672 pixels of 9 by 9 µm² (36 x 24 mm² active area) with a clock speed of 20 MHz. It is

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equipped with a very thin Gd2OS:Tb scintillator, making it inefficient for high-energy X-ray photons. Alternatively, a RadEye CMOS detector can be employed (Teledyne DALSA Inc., Ontario, Canada) which has a thicker scintillator, making it better suited for the analysis of highly attenuating samples such as geomaterials. The magnification of the object can be altered by changing the sample-to-source distance. The short experimental run time (~ 10 min) on this end-station makes it extremely well-suited as first step in the cascade of a multimethodology analysis on the scanner.

Figure 1 Image of the central µCT station, the W tube can be seen on the left, a sample is mounted on the motor stack in the middle and both X-ray detectors are visible on the right.

Confocal XRF The confocal XRF setup (Figure 2) can be employed for conventional 2D XRF experiments, 3D confocal XRF analysis and experiments using both techniques simultaneously. The setup contains a 50 W Mo anode tube equipped with polycapillary optics (Xbeam, X-ray Optical Systems, USA) to generate a focussed X-ray beam with a working distance of 3.6 mm and a spot size of 10 µm x 13 µm (H x V) at the Mo Kα – line energy (17.48 keV). Two large area SDD detectors (SiriusSD, SGX, UK) with 100 mm2 crystal area, which are positioned on either side of the source under a 90° angle, are used to collect the fluorescent photons. One of these detectors is employed in conventional 2D XRF-mode, while the other is equipped with polycapillary optics (XOS). By aligning the focal points of the X-ray source and the confocal detector equipped with these optics, a single micrometer-scale voxel can be analysed. The size of this voxel was determined to be approximately 30x30x30 µm³ using line scans over a 10 µm stainless steel wire (Goodfellow, UK). By moving a sample on a line or grid of such voxels, it is possible to extract direct 3D information. Due to the sensitive nature of the components of this setup and the time-consuming alignment procedure, it was opted to incorporate a laser-triangulation based positioning and safety system using the scanCONTROL 2D laser scanner (micro-epsilon, Oldenburg, Germany). This setup is ideal when the elemental composition of a few specific points of interest in a sample needs to be determined, since these points can be analysed directly and in a relatively short time span (~ 10 min/point). However, when elemental maps of larger areas of the sample are the aim of an experiment, measurement times can rapidly increase to several hours or even days.


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Figure 2 Picture of the confocal XRF setup, the Mo tube equipped with polycapillary optics is located in the center, the conventional detector on the left and the confocal detector (with optics) on the right. On the right the laser triangulation safety/positioning system is also visible.

XRF tomography In XRF tomography, line-scans are performed through the sample at multiple rotational angles. Analysis of the XRF spectra generates sinograms for all detectable elements present in the investigated object. Through the application of a suited reconstruction algorithm, a virtual slice of the scanned object can be retrieved.11,14,15 A Mo anode source with monocapillary optics (X-Ray Optical Systems) is used to generate the pencil beam which is needed for the XRF tomography procedure (Figure 3). The spot size of this beam is 20 µm in both vertical and horizontal direction (@ Mo−Kα, 17.48 keV) at 3 mm from the tip of the optics. The beam has a small divergence of 8 mrad. Two large area (170 mm²) SDD detectors (SGX) are employed in dual detection mode, maximizing the detector solid angle to nearly the full orb encircling the sample. The XRF tomography is the technique of choice when the 3D elemental distribution in a relatively large sample needs to be determined. Although the measurement time can still arise to several days for samples with a dimension of a few millimeter, this methodology is significantly faster than the confocal XRF analysis.

Motor system The heart of the experimental setup is the high-precision motor system, consisting of thirteen decoupled motors, installed on a large granite table (LAB, Leuven, Belgium). Additionally, the table has air-bearing controlled damping (Thorlabs, Newton NJ, USA) to minimize external vibrations. The motors are used for sample alignment, measurement and detector movements. An ultra-high precision air-bearing xyz-motor system (LAB) is used for the movement of the sample stage between the three different stations of the instrument (µCT, XRF-CT and cXRF). This stage has an extraordinary good reproducibility of ± 100 nm. These motors make it possible to perform the measurements in the same coordinate system at all three measurement stations. The rotational motor (LAB), crucial to the tomography techniques, is integrated in this motor system. On top of the rotational stage are two piezo motors (SmarAct, Oldenburg, Germany) which are employed to position the samples relative to the rotational axis of the system. Depending on the envisaged measurement, both the air-bearing and piezo motors can be used when scanning an object with one of the X-ray techniques. Finally, all detectors are motorized to some degree. A long range translational motor (LAB) allows the operator to select the most favorable detector for his or her µCT application, while smaller translational motors (LAB) are used to optimize the detector-sample position of the conventional XRF detectors (XRF-CT and part of the cXRF setup) to achieve the largest possible detector solid angle. The XRF detector equipped with polycapillary optics is installed on an xyz stage which is crucial in aligning the cXRF setup. Concept of the scanner Previous attempts at integrating multiple X-ray based analysis techniques into a single instrumental setup had a fixed sample stage with all components arranged around it. This allows for a compact setup, but unavoidably forces some compromises to the design. Therefore, the Herakles 3D X-ray scanner uses a fundamentally different approach, where the sample stage is no longer a fixed point in the setup. On the contrary, the high precision xyz motor stages allow movement of the sample stage from one analysis station to the other. The sample is aligned with the rotational axis using piezo electric motors on top of these main motor stages, which causes the sample coordinate system to be unchanged when translating it between the end stations of Herakles (Figure 4).

Figure 3 Picture of the XRF-CT station, the Mo tube equipped with monocapillary optics is in the middle, with two motorized, identical large-area SDD detectors to optimize the detector solid angle.



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two scans, coupling the data sets becomes relatively easy and straightforward.

Test pattern for the XRF stations Copper structures on a Si wafer were obtained by the following procedure. TiW (50 nm) and Cu (1 µm) were deposited on a p-type Si wafer by DC magnetron sputtering in the same vacuum cycle in an Alcatel SCM600 system. The Cu was patterned via photolithography and wet etching. A positive photoresist (Microposit S1818, Dow Electronic Materials) was applied on the sputtered wafer through spin coating (4000 rpm, 60 s) and soft baked. Next, the substrate was UV exposed (365 nm, 5.8 s at 14 mW/cm²) through a custom designed glass mask with a mask aligner system (SET MG1410) followed by development and hard bake. The exposed copper was etched in a Mecbrite CA-92 (Mec Europe) solution for 22 s and thoroughly rinsed in DI water. Subsequently, the TiW layer was etched in a 30% hydrogen peroxide aqueous solution at 55°C for 20 s, 10 s rinsed in 30% hydrogen peroxide aqueous solution at RT and a final rinse step in DI water was performed. Finally, the masking photoresist was stripped using acetone. The entire experiment was performed in a clean room (CR class ISO7 and ISO6). Daphnia magna model organism The Daphnia magna organism was cultured (monoclonal stock culture) at the Laboratory of Environmental Toxicology of Ghent University as described by Muyssen et al.31 The daphnia samples were prepared using a hexamethyldisilazane (HMDS) drying method. The samples were dehydrated in graded acetone solutions and then immersed in HMDS, which was left to evaporate overnight. The same sample preparation was previously used by De Samber et al. for their SRbased experiments on daphnia samples.32 Figure 4 Schematic overview of the instrument, from top to bottom: cXRF, µCT and XRF-CT. The image gives a nice view on the motor system movements connecting the end stations.

Since the three end-stations (µCT, XRF-CT and cXRF) are completely independent entities in this instrument, their components and settings are optimized for the envisaged methodology without compromising the performances of the other techniques. Hence, each single one of them is the state-of-theart of current lab-scale XRF and µCT technology. Furthermore, the segmented setup of the scanner permits a relatively easy change of components. This is an important asset, which allows upgrading the scanner with new components or testing of novel sources and/or detectors. A typical experiment on the 3D X-ray scanner starts at the centrally positioned µCT station, where a high-resolution three-dimensional image of the scanned objected is captured. Based on this image and the specific research question, our home developed software allows the user to select certain points, lines, areas, slices or even the complete sample to be investigated for their/its elemental composition using one (or both) of the XRF end-stations. The control software then autonomously moves the sample to the right position, taking into account the optimal angle-of-attack for the confocal imaging station. Since the coordinate system is retained between the

Perlite biofiltration support material Perlite is a naturally occurring siliceous volcanic glass which has the property to drastically increase in volume when it is rapidly heated to temperatures around 1000 °C. 33,34 In its expanded form it has several industrial applications in a wide range of sectors including construction, agriculture and the chemical industry.33-36 Its value as support material for heterogeneous catalysis has been demonstrated in the past.34 In the field of waste gas treatment biological techniques (biofiltration, bioscrubbing and biotrickling) are considered to be an effective and efficient choice for the removal of volatile organic compounds (VOC) from a gas flow, since the microorganisms used in these processes transform the VOC in small molecules (water, CO2, etc.), energy and biomass.37 The support material of a biofilter, on which these micro-organisms grow plays an important role in the biofiltration process. Amongst others it has to give support for the biofilm, yield a high contact area and provide the necessary mechanical strength. In this context, expanded perlite is probed as possible support material. During this study, preliminary feasibility tests for combined µCT/3D XRF scans as monitoring procedure for the biofilm were performed.


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Instrument characterization and test cases Characterization experiments To determine the analytical capabilities of the two XRF methodologies incorporated in the Herakles scanner, several experiments on reference materials with widely different matrices have been performed. These measurements are square 10 by 10 points mappings with 10 s dwell time per point. Adding the obtained spectra yields a 1000 s sum spectrum, averaging a relatively large area of the reference material, which is then analyzed and used to determine the detection limits of the setup. Using the NIST SRM 1577c bovine liver standard, the limits of detection for the XRF stations have been calculated according to the formula:

order to ameliorate the visibility of the features. The central image is made using the polycapillary source, while the lower one is obtained on the XRF-CT setup. Although rather vague, even the smallest line (2 µm) is still visible with the polycapillary source, this gives an idea of the smallest features which can be detected with this end-station (under optimal conditions) which is considerably smaller than its beamsize of about 10 µm. The scan on the XRF-CT setup only resolves the lines starting at 16.5 µm, which is approximately the beamsize (i.e. 20 µm) of its monocapillary source. Since the copper lines have a thickness of only 1 µm, the pattern is less suited for analysis of the confocal XRF measurements, because the depth of information and spatial resolution in the z-direction are in the range of 30 µm.

3 ∙ Where LODi, Ci, Ii and Ib represent limit of detection, certified concentration and measured intensity of element i and background intensity, respectively. In order to obtain comparable results, the corresponding Xray tubes were operated under the exact same conditions of 40 kV and 0.5 mA. Under these circumstances, the XRF-CT setup in dual detector mode and the confocal station in conventional mode have comparable limits of detection, being around 130 ppm for P, about 6 ppm for K and down to 2 ppm for Fe. Due to the much lower count rate detected with the detector equipped with polycapillary optics, the limits of detection of the confocal setup lie somewhat higher, at 56 ppm for K and 13 ppm for Fe.

Figure 5 Limits of detection calculated from a measurement (1000 s) on NIST SRM 1577c for the XRF techniques: confocal XRF (red ●), conventional XRF with the polycapillary source (blue ■) and dual-detector XRF-CT (green ▲) In cooperation with the center for microsystems technology (Ghent University and imec), a copper test pattern was designed and produced, enabling a profound assay of the spatial resolution of the two XRF techniques. The test pattern consists of different copper structures on a silicon wafer, which are produced by classical photolithography (as explained above). The main features of the structure are copper lines with variable width and interspacing (ranging from 100 µm to 2 µm), the exact dimensions of the lines were determined using optical microscopy as shown for the smallest lines at the top part of Figure 6. By investigating at which line width the instrument can no longer resolve two adjacent lines, a fair estimation of the spatial resolution of this technique can be obtained. The bottom part of Figure 6 shows the results of a scan with 1 µm step size over the test pattern, please note the image is enlarged in its vertical direction compared to the real x/y ratio in

Figure 6 Cu test pattern. (a.) microscope image used to measure the dimensions of the pattern, showing the smallest lines and CuKα maps of XRF scans on the conventional cXRF (b.) and XRFCT (c.) setups (1 µm step size, 10 s live time, 40 kV, 0.5mA) The zoom-in image shows the section with the smallest lines (2 µm – 20 µm), clearly showing the superior results with the cXRF setup compared to the XRF-CT setup.

Experimental case I: Daphnia magna An experiment using Daphnia magna as a model organism was performed to demonstrate the power of the new Herakles 3D X-ray scanner when analyzing the interior morphology and elemental composition of biological samples. First the three dimensional morphology of the sample was visualized using µCT. Figure 7 represents a rendering of the Dapnia magna sample, clearly showing some eggs and internal organs. Since the sample was relatively large (≈ 4mm high) a three dimensional scan of the complete organism using the 3D XRF techniques would have been very time consuming (timescale ≥ 1 week). Hence, the choice was made to investigate one virtual slice of the sample for its elemental composi-



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tion with the confocal XRF setup. In Figure 7 the position of this slice is indicated by a red dotted line.

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Figure 8 Combination of µCT and RGB (Fe, Ca, K) representation of the XRF data showing a virtual slice (for exact location see fig. 7) of the cXRF analysis (30 µm step size, 10 s LT, 40 kV, 0.8 mA)

Experimental case II: perlite support material

Figure 7 Rendered image of 360° µCT scan on the model organism (Daphnia magna) with the main visible organs named (1501 projections, 500 ms exposure time, 70 kV, 3W); the dotted line indicates the position of the XRF virtual slices.

Using the automated sample transfer from the µCT stage to the confocal XRF end-station, a mapping was performed with 30 µm steps. Figure 8 shows an RGB (Fe-Ca-K) image of the confocal XRF measurement, overlaid with the corresponding µCT slice. As can be observed, the slice visualized via confocal XRF analysis is exactly the slice selected using the rendered µCT measurement. This measurement clearly demonstrates the possibility of the instrument to selectively choose a specific region of interest with the µCT setup and analyze it for its 3D elemental composition. The Ca signal nicely outlines the organism, due to the calcium-rich structure of its carapax. Internally, some organs also show an elevated Ca content. K is mainly present in the internal organs of the daphnia, with an elevated presence in the eggs. Furthermore, Fe is also predominantly present in the eggs and some internal structures of the organism. In a comparable study using synchrotron radiation, De Samber et al.32 found similar elemental distributions for calcium and iron but they did not report on potassium.

Next to biological samples, as demonstrated with the first experimental case, the Herakles 3D X-ray scanner can serve a wide range of other possible application. The second test case deals with geological material (perlite) which is used as support material for biofiltration. These experiments were a proof of concept for the feasibility of the combined µCT/3D XRF methodology in the context of analyzing the structure and composition of the perlite support material and monitoring the active biofilm in these materials. The experimental procedure for these measurements once more started with a µCT scan to visualize the internal morphology of the samples. The top image of Figure 9 gives a virtual slice of the reconstructed µCT scan of one of the expanded perlite particles. It clearly demonstrate the open internal structure of these materials. Of special interest was the bright spot in the center of the image. Using XRF tomography, the elemental composition of this inclusion could be determined. The results of the XRF-CT scan are given as a reconstructed RGB image in the bottom part of Figure 9. Fe is indicated in red, K as a second element in blue and to visualize the internal structure, the Compton scatter is used with satisfactory results when compared to the µCT. The bright spot on the µCT image proved to be an iron rich inclusion with a diameter of about 40 µm present in one of the large voids of the expanded perlite structure. This second experimental test case amply demonstrates the added value of combining the two tomographic techniques. The low experimental time for the µCT (< 15min) allows for fast imaging of the entire sample with high resolution. Next to the valuable information on the sample extracted via this technique, it also allows for the selection of specific virtual slices of interest (e.g. where inclusions are present) to analyse for their elemental composition using a 3D XRF technique such as XRF-CT, which takes a lot longer (several hours up to one day). Hence, the total experimental time is minimized, without loss of information.


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Analytical Chemistry The test pattern gave direct information on the smallest features which could be imaged using the XRF techniques, proving the Mo-tube equipped with polycapillary optics is able to distinguish details down to a few microns, while the resolution of the XRF-tomography end station lies around 20 µm. The experiments on the Daphnia magna organism demonstrated nicely the ease of data integration with the new scanner and the advantages the combination of these 3D imaging techniques yield. Based on the high-detail µCT maps, the internal organs of the organism could be discerned, amongst others some eggs were clearly visible. Overlaid with XRF images, elevated elemental content could be allocated to the different structures. Ca was found to predominate in the carapax of the daphnid, while Fe and K were found in the eggs. The scans on the expanded perlite particle demonstrated the strength of the combined tomographic techniques (µCT and XRF-CT) to clarify the origin over certain features found during the µCT experiments such as the bright spot in Figure 9A which appears to be and iron rich inclusion. Furthermore, the information on X-ray attenuation yields a clear images of the complicated internal structure of these support material particles and could be used in a further study to identify the distribution of the biofilm in the pores of the perlite. As a test case for the feasibility of X-ray analysis of this type of materials these experiments were hence a great success.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interest.

Figure 9 (a.) µCT image (1501 projections, 500 ms exposure time, 70 kV, 3W) and (b.) XRF-CT RGB (Fe, Compton, K) image (20 µm step size, 10 s LT dual detector mode, 360° rotation, 90 projections 40 kV, 0.8 mA) of the same virtual slice through the perlite particle. The main structural features and the iron hot spot can be recognized in both images.

ACKNOWLEDGMENT The setup was developed within the framework of a Hercules-II project (AUG11-024). B.L. acknowledges the support by the IWT (agentschap voor Innovatie door Wetenschap en Technologie, Flanders, Belgium). The Special Research Fund of the Ghent University (BOF) is acknowledged for the post-doctoral grant of M.N.B.

REFERENCES

Conclusions Integrating µCT and element sensitive 3D XRF techniques into a single laboratory instrument proves to be a powerful and elegant tool to optimize the information gathered with each technique due to their complementary nature. The design of this instrument, where the sample is moved with ultra-high precision between the three independent end-stations, largely circumvents the spatial limitations more compact instruments put on each technique. Hence, the µCT, XRF-CT and confocal XRF end stations are separate state-of-the-art laboratory instruments, with very good resolution and high sensitivity. Furthermore, updating the setup with new components and features is much easier due to its modular concept. The strength of the instrument was demonstrated using three case studies, one on a micron level copper test pattern, the second on the biological model organism Daphnia magna and the last on expanded perlite support material for biocatalysis.

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