Daphnia magna is a freshwater crustacean and model organism for toxicology. Daphnia magna were fed with TiO2 anatase nanoparticles, fixed, embedded and sectioned according to routine electron microscopy protocols and correlatively investigated by secondary electrons (SE), scanning transmission helium ion microscopy (STHIM) and secondary ion mass spectrometry (SIMS). Since SE imaging gives information about the sample surface, no information of biological tissue can be retrieved from flat sections, but TiO2 anatase nanoparticles localized in the gut of the animal can still be visualized due to their intrinsic structure. Using STHIM, biological structures of the gut epithelial cells, such as Villi (V), cell borders (arrows) and the basal labyrinth (bas lab) can be readily identified (top row). STHIM therefore facilitates recognition and orientation of biological structures in flat sections that are inaccessible to SE imaging. The middle row shows correlative high resolution SE and SIMS imaging of the TiO2 nanoparticles – epithelium interface: While SE imaging just reveals the TiO2 nanoparticles, SIMS allows the detection of CN- cluster-ions (as a marker for biological tissue) and the detection of jTi+ + kTiO+ ions (i.e. all isotopes of Ti+ and TiO+ in order to increase dynamic). The obtained chemical maps can be superimposed, and false colour coded to identify co-localizations of secondary ion-species, allowing the precise localization of nanoparticles in biological tissues. The bottom row shows mass spectra obtained in positive and negative SIMS mode from the region of interest presented in the middle row, highlighting the benefit of the focal plane detector, as a multitude of secondary ions can be investigated at the same time in a chosen mass range.
As proof of concept system, TiO2 E171 nanoparticles (NPs) have been investigated in secondary electron (SE) and secondary ion mass spectrometry (SIMS) mode. The SE image (top left), obtained using the He+ primary ion beam, highlights the fact that the NPs tend to agglomerate in larger but also smaller clusters (white arrow). To find a single NP/very small clusters of NPs is rare, nevertheless as highlighted by the red arrow it is possible to also image these features in SE mode. The mass spectrum (top right image), obtained in positive SIMS mode using the Ne+ primary ion beam, demonstrates clearly the advantage of the novel continuous focal plane detector by being able to record in parallel all mass peaks over the selected mass range. In the case of TiO2 NPs it allows to have access to all titanium isotope peaks as well as all related titanium oxide peaks at the same time. In this manner for SIMS imaging it is possible to increase the dynamic (counts level) within such an image, as several SI signals can be added up representing the same feature in the sample. Here, the bottom left image represents the 48Ti+ peak signal alone, the central bottom image groups all titanium isotope peak signals (jTi+) and the bottom right image adds on top of those also the signal from all titanium oxide peaks (jTi++ kTiO+). Moreover, the single NP/very small cluster of NPs is also detectable in the SIMS mode (red arrow).
The EU funded COST network project “Focused Ion Technology for Nanomaterials - FIT4NANO”, which was initiated by the Helmholtz-Zentrum Dresden-Rossendorf, was created to enhance the collaboration between European universities, research institutes and companies to develop new technologies and applications that go beyond classical applications in materials science. The focus will be on next generation nanotechnology, including future medical applictions. The collaborations will be organised in four groups: i) the development of future technologies, ii) its application to nanostructured functional materials, iii) ion - solid interactions, and iv) communication and public relations.
For more information visit the website of the HZDR.
The general assembly at month 48 was organised as a video conference to discuss among the 9 partners the progress of the npSCOPE project after the initial 48-month duration of the project and to prepare the work remaining for the project extension of 6 months.
Right before Christmas 2020, the motorised part of the newly developed microscopy stage was received at LIST. Onto this setup a specialised cryo-microscopy table is installed that can hold typical microscopy stub sample holders as well as transmission grid holders. Bundles of copper bands are on one side attached to the microscopy table and will under vacuum on the other side be attached to the cold finger of the liquid nitrogen dewar in order to cool it down to a very low temperature of about -140° C. A test chamber is set up and ready for stage movement and cooling tests under vacuum prior of integration into the npSCOPE instrument. The developed stage setup is the important last part for the overall cryo-preparation/investigation workflow.
At the beginning of December 2020 the second part in order to establish cryo-workflows at LIST has arrived, namely the cryo-glove box. As can be seen, on its left side panel it is equipped with a docking station to attach the cryo-transport suitcase. At the inside a small vacuum chamber with internal cooling station and a grabbing rod is used to pull sample holders out of a liquid nitrogen cryo-preparation bath back up into the vacuum chamber. Within the chamber the atmosphere is exchanged from nitrogen gas to vacuum prior of loading then the cryo-cooled sample holders into the attached transport suitcase.
In September 2020 the ultra-high vacuum (UHV) cryo-sample transport suitcase arrived at LIST. This system represents a first item in order to establish high quality cryo-sample preparation and transfer workflows. The suitcase, equipped with a specialised compact vacuum pump, easily achieves a base vacuum level in the range of 1e-10 mbar or even below and can transport samples at temperatures below -140°C. It will be used in order to bring samples from a cryo-preparation glove box to the npSCOPE instrument loadlock under vacuum and cold conditions, before transferring in a last step the cryo-samples into the microscope chamber.
The general assembly at month 42 was organised as a video conference to assess the situation of the npSCOPE project at the end of the lockdown period, to coordinate the restart of the laboratory activities and to plan the work for the six months to come.
Due to the delays caused by the COVID-19 lockdown, the European Commission has granted a 6-month project extension to the npSCOPE project. Major activities affected by the lockdown include the toxicology studies and some hardware developments for the npSCOPE instrument.
The general assembly at month 36 was organised by partner MJR PharmJet GmBH in Überherrn, Germany. During two days, the progress of the different work packages was discussed and the work for the remaining 12 months was planned. The General Assembly finished with a visit of the facilities of MJR PharmJet.
The Helium Ion Microscope (HIM) has been installed and made available to partner FAU through INAM GmbH (Forchheim, DE) and is now operational. First images show TiO2 nanoparticles in the gut of a daphnia. The next step will be the installation of the mass spectrometer on the HIM.
The focal plane detector of the npSCOPE instrument is now fully operational. Below are shown some images of major components in a Perovskite solar cell.
The second review meeting of the npSCOPE project was organised at LIST in Luxembourg to present the progress of the project, in particular the assembly of the npSCOPE instrument, and to make some demo of the npSCOPE instrument to our Project Officer from the European Commission and our external reviewer. The project was evaluated very positively and helpful suggestions on project implementation and the interaction with other European projects were received.
The first cryo-components have been installed within the main chamber of the npSCOPE instrument. A large cryo-shield now covers the interior ceiling of the analysis chamber and is connected to a liquid nitrogen dewar. First cool down/warm up cycles have been successfully performed.
The detector for Scanning Transmission Ion Microscopy (STIM) was installed into the bottom pumping tube of the main chamber of the npSCOPE instrument. To adjust its position, it has been mounted on an extension arm.
The general assembly at month 30 was organised by the partner from the ETH in Zürich, Switzerland. During the first day, the progress of the different work packages was discussed and the review meeting at the end of June was prepapred. During the second day, the progress of the npSCOPE project was presented to the Advisory Board which led to fruitful discussions and to advice on project implementation.
Applications beyond nanotoxicology are being explored. Partner FAU has prepared samples of microsized GaN rods doped with Si (lower part) and Mg (upper part) (cf. figure) for npSCOPE correlative microscopy analysis. As another application for the semi-conductor industry lithium distribution in next-generation batteries prepared by added manufacturing is of interest.
The potential of correlative microscopy combining high-resolution structural and chemical information will also be tested for medical purposes, for example to detect the spatial distribution of 12C15N (compared to 12C14N) after 15N-thymidine incorporation into the DNA, or accumulation of fluoride from drinking water in tissue biopsies from intestine, liver, heart and/or brain.
The main vacuum chamber of the instrument has been installed onto the anti-vibration frame at LIST with the support of ZEISS. Currently, the instrument is equipped with the GFIS column, the ET detector, the electron flood gun, the standard NanoFab load-lock as well as the standard NanoFab stage. A test phase to assess if the specifications of the GFIS column are met will be started now.
Such a study have been performed to provide samples of interest for correlative imaging using the npSCOPE instrument. Partner TOXALIM showed no major impact of 22 nm TiO2 (P25, Degussa) on barrier integrity and no evidence of DNA strand breaks in human buccal epithelial TR146 cells cultured in vitro on filters and exposed for 24 hours to exposure concentrations up to 100 µg/ml. However, a dose-dependent chromatin remodeling was observed, which could be decreased again when the nanoparticles were washed out after 2 hours exposure. Whether the TiO2 nanoparticles are directly and physically involved in altering the chromatin structure in the nucleus will be further investigated through combined TEM and HIM-SIM analyses of the exposed cells.
Partner LIST has performed benchmarking HIM-SIM analyses on its 3D co-culture model of the small intestine (Caco-2/TC7 gut epithelial cells combined with HT29 mucus-secreting goblet cells) exposed to TiO2 nanoparticle after in vitro simulated digestion (cf. Figure).
The GA meeting at month 24 was organised by the partner from the Friedrich Alexander Universität in Forchheim, Germany.
During the two days progress in the different work packages was discussed and objectives for the next six months were set. The meeting finished with a visit of the local laboratories.
Zeiss was organising in collaboration with LIST the 1st European Orion Nanofab SIMS Workshop where they introduced the new system combining ultimate high resolution imaging with chemical information.
This workshop took place on 4-5 December in Luxembourg. The event included presentations from internationally leading researchers covering high resolution SIMS applications in the fields of material science, life science and geology. Discover new trends in high resolution materials analysis whilst getting hands-on practical experience with this new technology.
More information can be found on the workshop website.
At the Helmholtz-Zentrum Dresden-Rossendorf, molecular dynamics computer simulations are employed to study the sputtering yield and channeling effects in Gold nano-clusters of different sizes. Primary ion energy and crystal orientation are varied to obtain a holistic image of the possible effects relevant for scanning transmission ion microscopy. Our results show that ion-channeling occurs not only in the principal low-index, but also in other directions in between. The strengths of different channels are specifed, and their correlations with sputtering yield and damage production is addressed. The figure below shows sputtering under 30 keV He ion irradiation of 5 nm Gold nano-clusters.
The GA meeting at month 18 was held in Oberkochen, Germany at the Zeiss headquarters. During the first day, the focus was on instrument design and assembly, cryo and correlative workflows and data processing. On the second day, project coordination and management were discussed.
A SIMS system has been installed on the test chamber with the GFIS column at LIST in Luxembourg in order to get first mass spectrometry data on the npSCOPE system:
During the tests, first images of a Li-TiO nanoparticle mixture could be obtained. The Li+ signal is shown on the left and the Ti+ signal on the right:
The Review Meeting and GA at month 12 was held in the Helmholtz Zentrum Dresden Rossendorf (HZDR) in Dresden, Germany.
During the first day the developments of the first year were reviewed. During the second day, specific tasks were discussed in more detail and the work for the 6 coming months prepared. The GA finished with a visit of the facilities of HZDR.
The GFIS source and column manufactured by ZEISS have been shipped to Luxembourg and are now installed on a test chamber in the npSCOPE laboratory at LIST. The specification tests for He+ and Ne+ beams were successfully fulfilled. The different npSCOPE partners are in the final stages of designing the various other components of the npSCOPE instrument (e.g. STHIM system, SIMS system, cryo-stage, main chamber, load lock system, ...). These components will be integrated progressively during the next months.
The new laboratory at LIST which is going to house the npSCOPE instrument is now ready!
At LIST, currently tests are being performed with a Z-X-Y-R piezo-stage when a set of copper bands is attached to it. These tests are needed to progress with the development of the actual cryo-transmission-stage for the npSCOPE instrument.
The constrution of the extension of the LIST building, which is going to hold the npSCOPE instrument, started today.
The GFIS column for the npSCOPE instrument is being assembled and tested at ZEISS.
The GA meeting at month 6 was held in Lake House near the VITO headquarters in Mol, Belgium.
During one day and a half, 21 members of the 9 project partners discussed the progress of the different work packages.
The kick-off meeting for the Horizon 2020-funded project npSCOPE was held in Belvaux in Luxembourg. It was hosted by the Luxembourg Institute of Science and Technology (LIST), the project coordinator.
During this 2 day event, 27 members from the 9 partner institutes were fixing the working programme for the first 6 months.