The presented scanning transmission x-ray microscope (STXM), build on top of our existing modular platform (FlexIX) for high resolution imaging experiments, allows versatile investigations of different samples. The FlexIX endstation allows to switch between a Full Field and a STXM mode. For the STXM mode we use a spatialy resolved detector together with an energy dispersive detector, this allows to investigate the morphology and the chemical or elemental distribution of the sample simultaneous. The combination of the nanoscopy endstation and the XUV beamline P04 results in a powerful tool for investigations of life science samples.BMBF, 05K13UL1, Verbundprojekt 05K2013 - Hochauflösende Bildgebung: Hochauflösende Bildgebung im EUV- und weichen Röntgenbereich bei PETRA III und FLASH. Teilprojekt 1

Andrianov, K.

Dierks, H.

Gnewkow, R.

Haidl, A.

Kanngießer, B.

Lötgering, L.

Lühl, L.

Nisius, T.

Wilhein, T.

Journal of Physics Conference Series

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1Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distributionof this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.Published under licence by IOP Publishing Ltd1234567890X-Ray Microscopy Conference 2016 (XRM 2016)  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 849 (2017) 012007  doi :10.1088/1742-6596/849/1/012007Scanning Transmission X-ray Microscopy with X-rayFluorescence Detection at the XUV Beamline P04,PETRA III, DESYK Andrianov1, L Lühl2, T Nisius1, A Haidl1, R Gnewkow2,L Lötgering1, H Dierks2, B Kanngießer2 and T Wilhein11 Institute for X-Optics, University of Applied Sciences Koblenz Rhein-Ahr-Campus,Joseph-Rovan-Allee 2, 53424 Remagen, Germany2 Institute of Optics and Atomic Physics, Technical University of Berlin, Hardenbergstr. 36,10623 Berlin, GermanyE-mail: andrianov@hs-koblenz.deAbstract. The presented scanning transmission x-ray microscope (STXM), build on top ofour existing modular platform (FlexIX) for high resolution imaging experiments, allows versatileinvestigations of different samples. The FlexIX endstation allows to switch between a Full Fieldand a STXM mode. For the STXM mode we use a spatialy resolved detector together withan energy dispersive detector, this allows to investigate the morphology and the chemical orelemental distribution of the sample simultaneous. The combination of the nanoscopy endstationand the XUV beamline P04 results in a powerful tool for investigations of life science samples.1. MotivationClassical optical microscopy techniques are fundamentally limited by the diffraction limit, whichimplies that spatial resolutions below 200 nm can only be achieved by the use of short-wavelengthradiation. While electron microscopy enables two- and three-dimensional imaging of materialsin the sub-nanometer range [1], there is a gap in spatial resolution in the 10th nm range, whichcan be covered with X-ray Microscopy (XRM). For state of the art investigations in biomedicinethis spatial resolution range is of a great interest [2]. In addition, the penetration depth of XRMis much higher as compared to electron microscopy, which facilitates sample preparation.The P04 beamline of Petra III is a variable polarisation XUV beamline with an energy rangeof 250 eV up to 3000 eV [3]. This energy range is suitable to identify most of the interestingelements typically found in biological samples [2]. The beamline provides exceptionally highphoton flux of up to 1012 photons per second in combination with a very high resolving power(E/∆E > 10.000) [3]. In contrast to most other beamlines, P04 has no fixed endstation and theexperimental setups are provided by the user. In order to perform XRM at the P04 beamlinethe variable FlexIX endstation was developed and tested in the laboratory.The FlexIX endstaion consists out of up to three separate vacuum chambers and custom de-signed high resolution piezo stages. These piezo stages are able to move precisely in x-, y- andz-direction, which makes it possible to adjust components for different imaging modes.21234567890X-Ray Microscopy Conference 2016 (XRM 2016)  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 849 (2017) 012007  doi :10.1088/1742-6596/849/1/012007Figure 1. Experimental setup of the scanning microscope. Top: Sketch of the setup, bottom:real setup insight the vacuum chamber. 1. Fresnel zone plate; 2. Order sorting aperture (OSA);3. Fluorescence detector; 4. Sample on scanner; 5. Phosphor screen; 6. CCDThe endstation is designed for quick changes between full-field (FF) mode and STXM/ fluo-rescence mode.The main advantage of FF mode is that a large area of the sample can be investigated withvery high spatial resolution ≈ 30 nm with a single exposure. During recent experiments theendstation was operated as a transmission x-ray microscope (TXM) in FF mode [4].First experiments with the STXM mode were done in October 2015 and June 2016. In STXMmode the spot size on the sample defines the spatial resolution of the system. Therefore a zoneplate (ZP) is used to focus the radiation on a small spot. Then the sample is moved throughthe focus point in a raster grid or in a continuous on-the-fly scan to retrieve an image.In STXM similar resolutions with respect to FF can be achieved, but since scanning is needed,investigations of large areas are quite time consuming. However, in contrast to FF differentimaging options such as phase-contrast or dark-field are easier to implement [5] and elementalcontrast by the detection of characteristic fluorescence radiation is accessible.2. Experimental SetupA sketch of the STXM setup is shown in Figure 1. X-Ray radiation from the beamline is focussedby a zone plate with 333 µm in diameter and an outer most zone width of 40 nm onto the sample.The zone plate consists out of 150 nm tungsten deposited on a 100 nm thick SiN membrane andpossesses a central stop of gold with 160 µm in diameter. Between ZP and sample is an ordersorting aperture (OSA) made of 100 µm thick stainless steel with a 150 µm hole. It is mountedon a small aluminium tube in order to prevent the fluorescence radiation from the OSA to reachthe detector.The sample is scanned by a P733 scanning stage from Physik Instruments (PI). It has a travelrange of 100 µm x 100 µm with a resolution of 0.1 nm.The sample holder is designed to have space for up to eight samples prepared on commontransmission electron microscopy (TEM) SiN membranes or grids.The detection of the transmitted intensity distribution is done indirect with a P43 phosphorscreen which is used to convert the X-rays into visible light. An optical path is used to transferthe visible light out of the vacuum chamber and an objective is used to image the signal fromthe phosphor screen on the Andor iXon3 EMCCD detector with 128 x 128 pixel and a pixel sizeof 24µm x 24 µm.31234567890X-Ray Microscopy Conference 2016 (XRM 2016)  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 849 (2017) 012007  doi :10.1088/1742-6596/849/1/012007Figure 2. Transmissionimage of a test pattern,taken at 720 eV photonenergy, smallest structures50 nm. On-the-fly scan, stepsize: 20 nm, field of view:5.4 µm x 5.4 µm, scan time:72 min. (without electronmultiplier)Figure 3. Mouse liver cells grown on a 200 nm thickSiN membrane, after air drying and fixation the sample wasinvestigated by 720 eV photon energy, top left: Light microscopeimage of the sample; top middle: Transmission image, top right:Fluorescence image, (red) iron, (blue) oxygen, (green) nitrogen;bottom: Fluorescence signal of (Fe) iron, (N) nitrogen,(O) oxygen; Step size: 1 µm, field of view: 100 µm x 100 µm,scan time: 34 min. (without electron multiplier)Using a spatially resolved detector for the transmitted intensity allows to gather several imagingmodes such absorption contrast and phase contrast simultaneously [5]. This is done withoutany change in the optical path only by different treatment of the experimental data from thetransmission detector. For each of the several imaging modes specific evaluation of the data isrequired to generate the differnet images.The STXM is also suitable to investigate the elemental distribution. Characteristic X-rayfluorescence radiation from the sample is detected by a windowless 30 mm2 SDD detector(Brucker Xflash 5030) with an enrgy resolution of < 127 eV FWHM @ MnKα.For each beamtime the endstation has to be assembled and aligned, this takes typically oneday. After the alignment procedure is completed, usually a resolution test is performed. Anexemplary STXM result is shown in Figure 2. The used test object was a Siemens star with50 nm smallest structures consisting of 200 nm thick layer of tungsten on a 100 nm thick SiNmembrane.During the last phase of the commissioning of the experimental setup, it was possible to performon-the-fly scans. This was a great and important step, since it reduces scanning time significantly.3. Biological imagingThe STXM setup was used for biological samples with interest to the transport mechanism oflipids through the blood circuit into adipose tissue.In first tests mouse liver cells marked with iron nano particless (SPIO) grown on 200 nm thickSiN windows were investigated. After air drying and fixation with 2.5 % formaldehyde, thesamples were investigated with the STXM endstation with a photon energy of 720 eV (Figure3). During the first beamtime the resolution was limited, so it was not possible to resolve theagglomerations of iron particles which are expected to be in the 100 nm range. Nevertheless,the combination of fluorescence and transmission microscopy is working, as can be seen in figure3.In the second beamtime tissue slices from brown adipose tissues of a mouse were investigated.41234567890X-Ray Microscopy Conference 2016 (XRM 2016)  IOP PublishingIOP Conf. Series: Journal of Physics: Conf. Series 849 (2017) 012007  doi :10.1088/1742-6596/849/1/012007Figure 4. Tissue slices from brown adipose tissues of a mouse. The tissue is marked with ironnano particles (SPIOs). The slices are ≈ 3 µm thick, placed on a 200 nm thik SiN membrane andfixed with 2.5 % formaldehyde. After air drying the samples were investigated with a photonenergy of 750 eV . Left: Light microscope image of the sample; Midle: Transmission image;Right: Differential phase contrast (horizontal); On-the-fly scan; 270 x 270 Pixel; Field of view:54 µm x 54 µm; Scan time: 72 min. (without electron multiplier)The ≈ 3 µm thick slices were deposited on 200 nm thick SiN windows (TEM windows) andwere fixated with 2.5 % formaldehyde. After air drying, the samples were investigated using aphoton energy of 750 eV . Some results are shown in figure 4.4. OutloockIn order to reduce radiation damages for biological samples, a cryo environment including atransfer system will be implemented. The bottleneck of the system with respect to fasterscanning times is the solid angle of the fluorescence detector. A new detector with approximatelyten times larger solid angle of detection will be implemented. For this detector a semi-quantitative approach for the analysis of the fluorescence data will be developed.AcknowledgmentsThis project is financially supported by the German Federal Ministry for Education and Research(BMBF) at the DLR (05K13UL1).References[1] Chen, Chien-Chun, et al. ”Three-dimensional imaging of dislocations in a nanoparticle at atomic resolution.”Nature 496.7443 (2013): 74-77.[2] Kirz, J., Jacobsen, C., & Howells, M. (1995). Soft X-ray microscopes and their biological applications.Quarterly reviews of biophysics, 28(01), 33-130[3] Viefhaus, Jens, et al. ”The Variable Polarization XUV Beamline P04 at PETRA III: Optics, mechanicsand their performance.” Nuclear Instruments and Methods in Physics Research Section A: Accelerators,Spectrometers, Detectors and Associated Equipment 710 (2013): 151-154.[4] Wessels, Philipp, et al. ”Time-resolved imaging of domain pattern destruction and recovery via nonequilibriummagnetization states.” Physical Review B 90.18 (2014): 184417.[5] Feser, M., et al. ”’Scanning transmission X-ray microscopy with a segmented detector.”’ Journal de PhysiqueIV (Proceedings). Vol. 104. EDP sciences, 2003.

Scanning Transmission X-ray Microscopy with X-ray Fluorescence Detection at the XUV Beamline P04, PETRA III, DESY

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Scanning Transmission X-ray Microscopy with X-ray Fluorescence Detection at the XUV Beamline P04, PETRA III, DESY

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