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Dear Professor, dear Scientist, dear Reader,
Thanks to the appreciated support of many of you, of ScopeM Steering Board, the scientific equipment program (SEP) of ETH Zurich, we were able to further extend the portfolio of microscopy techniques and methods available at ScopeM for its broad users’ community.
Those include notably a new confocal spinning disk combined with a super-resolution microscope "Nikon SoRa", a Quantitative Phase Imaging system, advanced optical tweezers installed on a Raman confocal, and analytical add-ons at the SEM Hitachi SU5000. You will also find two short contributions on novel applications in the field of Liquid Phase TEM for in-situ electro-chemical studies of transition metals, as well as the study of metallic nano-printed pillars made by a technique coined “electro-hydrodynamic redox printing” as developed in D-MATL’s Laboratory for Nanometallurgy. The nano-printed pillars were characterized by multiple electron and ion beam techniques – including with Atom Probe Tomography - at various orders of magnifications.
I hope you enjoy this issue of ScopeM newsletter and wish you much success in your further research, teaching and projects.
Best regards,
Nicolas Blanc, PhD
Managing Director, ScopeM |
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| Nikon SoRA |
ScopeM recently acquired a combined confocal/super-resolution microscope based on the ''Optical pixel re-assignment principle''.
The new system is basically a confocal Nipkow spinning disk, equipped with the latest Yokogawa Confocal Scanner Unit CSU-W1-T2 SoRa (Super resolution via optical Re-assignment). It contains two interchangeable spinning disks, one with micro-lenses before the emission pinholes and a magnification adjustment. The combined micro-lenses/pinholes on the SoRa disc mimic the effect of an infinitely small and ideal pinhole, but not compromising signal brightness.
Due to the new disk design the system almost reaches the z-resolution of a point scanning confocal. It is equipped with two back illuminated sCMOS Hamamatsu Orca Fusion BT cameras (2304x2304 pixels, 6.5x6.5 um pixel size) that allow large field-of-views as well as fast and sensitive live-cell imaging even in a dual camera mode.
In addition, it is equipped with a point scanner for FRAP for doing precise FRAP and ablation experiments. It has a full box incubator for temperature and CO2, a piezo z-stage (Mad City Labs Nano-Drive, 200 µm, with triggering) and filter combinations for fast multi-color acquisition. It has also a hardware autofocus (Nikon Perfect Focus System). The two sCMOS cameras allow for simultaneous acquisition of combinations of blue and yellow, green and red, and blue and far-red fluorophores.
Furthermore, the system is equipped with another optical pixel re-assignment imaging microscope, using the new technology of Re-scan with the RCM system from confocal.nl.
The Nikon SoRa is ideal for fast, live cell imaging. Super-resolved images down to 120nm lateral resolution are acquired optically, on any sample, without special preparations or computational steps.
Main contact: Dr. Dora Pinotsi (email) and Joachim Hehl (email) |
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| Quantitative Phase Imaging - Tomocube |
With the new quantitative phase imaging (QPI) system of Tomocube ScopeM has expanded its regime of label free imaging technologies such as Raman Spectroscopy, CARS/SRS (providing chemical contrast), SHG (collagen visualization in tissues) by yet a new contrasting approach: Tomocube 2HT-"H QPI
QPI offers contrast based on differences in Refractive Indices within transparent biological samples allowing for visualization of compartments of different optical densities within cells and tissues. Using a holographic approach by collecting a series of RI holograms from 360° around the sample it provides phase information in 3D with a maximal spatial resolution of 110x110x220nm and a temporal resolution of 400ms. Due to the linear correlation of RI to protein concentration this technique can extract quantitative data such as volume, surface area and dry mass, rendering it useful for applications such as phase separation, bacterial growth or apoptosis without the need of any labeling. Fully incubated for temperature and CO2 and equipped for automatized time lapse and multi positioning acquisitions the system enables kinetic and long-term studies in living cells under low phototoxic conditions. In addition, it is equipped with a fluorescence module allowing for phase and fluorescence image correlation. Currently the system can be used “only” with a high NA (1.2) water immersion objective (relatively small field of view) but during the 1st half of 2022 the arrival of a second system is scheduled (for lower magnification/resolution but higher throughput applications).
Main contact: Dr. Sung Sik Lee (email)
For further reading
https://www.tomocube.com/technology/holotomography/
https://www.tomocube.com/applications/
https://www.pnas.org/content/118/31/e2103956118.short?rss=1
https://www.nature.com/articles/s41556-021-00802-x |
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| Optical Tweezers |
A new optical tweezers setup is installed at the Horiba Raman confocal microscope of ScopeM.
We have purchased a CellManipulator system from Molecular Machines & Industries (MMI) and the setup is installed on a inverted microscope (Nikon Ti-E with an incubator for biological applications). This optical tweezers system is based on the mechanical forces arising from a strongly focused laser beam (wavelength: 1070nm, 10W continuous wave). It offers up to 20 traps simultaneously (with time sharing) with two independently controllable beams. Thus, it can hold multiple microscopic objects and move simultaneously and independently. Further, it is coupled to a quadrant detector (QD) that enables to quantify the force applied on an object hold by the tweezer (e.g. adhesion force between the attached cells by holding and modulating the relative position between trapped cells). The system can be used for wide-field transmission/fluorescent imaging and also in combination with Raman microscopy. Hence, it opens up opportunities to perform various interdisciplinary projects combining biology, chemistry, material science etc.
Detailed description of the system and application can be found under:
https://www.molecular-machines.com/products/cellmanipulator
https://www.molecular-machines.com/blog/new-application-note-optical-trapping-and-force-spectroscopy-of-non-spherical-rod-shaped-bacteria-and-diatoms
Main contact: Dr. Sung Sik Lee (email). |
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| Analytical add-ons (EDS and EBSD) installed at the SEM Hitachi SU5000 |
In February 2022, Oxford Instruments installed new detectors for energy dispersive spectroscopy (EDS) and electron backscatter diffraction (EBSD) at the Hitachi SU5000 scanning electron microscope (SEM). This complete analytical system was acquired through an ETH-SEP grant and broadens the fleet of analytical SEMs in operation at ScopeM. The hardware is composed of two large area window (100 mm2) Oxford Ultim Max 100 EDS detectors mounted at opposite sides to the column, and the Oxford Symmetry S2 EBSD detector. The benefits of two EDS detectors are: (i) It decreases considerably the shadowing effects in EDS mapping on rough surfaces; (ii) It doubles the amount of X-ray counts detected for the same electron beam; (iii) It provides redundancy in the unfortunate case one of the detectors is not working. The Oxford Symmetry S2 EBSD detector has CMOS technology with a maximum pixel resolution of 1244 x 1044 and can operate at a maximum speed of 4500 frames per second. Therefore, it can be used for collecting high quality Kikuchi diffraction patterns as well as for fast crystallographic orientation mapping.
Further information:
https://nano.oxinst.com/products/sem-and-fib
https://nano.oxinst.com/symmetrys2
https://nano.oxinst.com/products/ultim-max
Main contact: Dr. Luiz Morales (email) or Dr. Peng Zeng (email). |
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| Figure: a) SEM Hitachi SU5000 with two EDS detectors and EBSD detector from Oxford Instruments; b) Elemental (top) and crystallographic orientation (bottom) maps simultaneously acquired on W-Ti carbides in a superalloy matrix (sample kindly provided for evaluation tests by Jona Engel, Laboratory of Nanometallurgy, ETH Zürich). |
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| Liquid Phase TEM: Electro-Chemical Studies of Catalytic Reactions of Water Oxidation |
Fundamental understanding of the molecular mechanisms of water-splitting reactions as sources of molecular hydrogen is very important for the development of renewable energy solutions. The performance of the water-splitting-based energy storage modules is limited by oxygen-evolution reactions (OERs) that in turn are mediated by transition metals based electro-catalysts. The understanding of kinetic and dynamic processes occurring during the OERs and the factors monitoring the activity of the catalysts is one of the research directions of the group of Professor Greta Patzke (University of Zürich, Department of Chemistry). The time-resolved electro-chemical response of the OER cells synchronized with the microstructure evolution data enables deep physical and chemical understanding of these catalytic reactions and the interlink between the reaction mechanism and microstructural changes. It provides unique information about the real oxidation pathways revealing the complex arrays of real-time data on electro-chemical dynamics at the electrode-electrolyte interfaces. This information is crucially required for the design of molecular catalysts.
Within the frame of the PhD work of Esmael Balaghi, the dynamic processes occurring during OERs mediated by synthesized catalysts of Mn, Co and Cu compounds are studied by a combination of complementary techniques such as SEM, EXAFS, XANES, TEM, as well as by time-resolved in-situ electro-chemical liquid-phase TEM (LPTEM). The latter was carried out in collaboration with ScopeM team on the in-situ JEOL Grand ARM 300F “Vortex” using the dedicated liquid phase in-situ “Poseidon” holder (Protochips). The reliable and simultaneously simple measurement set-up is schematically shown at the bottom of the Figure.
One of the studies concentrated on the Cu-based catalysts. The sequence of TEM micrographs in (a) presents snap-shots of the early stages of the nucleation and growth of the copper oxide particles (bright yellow) at the surface of the working electrode (blue). The phase reaction at the electrode – electrolyte interface verified by electron diffraction patterns (eDPs) analyses (b, c) and by spectrum imaging (d, e) confirms the formation of CuO phase in the electrolyte. The studies revealed the actual OER mechanism in the presence of the Cu-complex catalyst is more complex than it was assumed and occurs through the formation of Cu oxide at very low potential before the actual onset of the OER. This strongly suggests that CuO particles are the true catalyst for the reaction.
Main contact: Dr. Alla Sologubenko (email) |
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Electro-chemical LPTEM studies of the Cu-complexes mediated OER.
(a) Degradation of the Cu-complexes during an anodic scan during cyclic voltammetry versus Ag/AgCl reference electrode. The sequence of TEM micrographs presents snap-shots of the early stages of the nucleation of the copper oxide particles (bright yellow) at the surface of the working electrode (blue). The phase state change of the liquid phase at the electrolyte is verified by electron diffraction pattern (eDP) analyses: (b) shows the eDP from the Pt-working electrode, whereas (c) demonstrates the presence of the CuO-phase particles. The elemental content analyses of the electrode adjacent region were carried out using energy-dispersive X-ray spectroscopy (EDS) in a spectrum image (SI) mode (d, e). The EDS SI confirms the formation of CuO phase in the electrolyte. The schematic of the CuO unit cell is shown in (f). The schematic of the electro-chemical in-situ TEM set-up in operation.
1“Mechanistic Understanding of Copper-based Homogeneous Water Oxidation Catalyst by In Situ Electrochemical Liquid Transmission Electron Microscopy”,
S. Esmael Balaghi, Somayeh Mehrabani, Younes Mousazade, Robabeh Bagheri, Alla S. Sologubenko, Yhenlun Song, Greta R. Patzke, Mohammad Mahdi Najafpour.
ACS Applied Materials & Interfaces, 2021, 13 19927-19937. https://doi.org/10.1021/acsami.1c00243 |
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| Movie caption: In situ electro-chemical liquid phase TEM (EC-LPTEM) analyses directly visualize the early stages of oxygen evolution reaction (OER) in the presence of Cu-complex catalyst. The OER is accompanied by growth of copper oxide particles at the surface of the working electrode |
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| The pH-sensitive nature of structural polymorphs of α-syn fibrils revealed by cryo-EM |
| The α-synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's Disease. The conversion from monomer to aggregate is a complex step and the aggregation rates depend on external conditions like, pH, temperature, buffer composition. At slightly acidic pH values, usually present in various intra-cellular locations, including endosomes and lysosomes, the aggregates multiply and coalesce much faster than at normal physiological pH values. This is largely a consequence of much more rapid secondary nucleation. In this study, the focus is structural elucidation of α-synuclein fibrils prepared at a pH of 5.8 in Phosphate-buffered saline (PBS) to replicate aforementioned physiological conditions. |
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The structural analysis revealed arrangement of two protofibrils of -synuclein at resolution, which allowed us to build the atomic model including amino acid side chains. The new conformation (gold; Figure D) resembles published polymorph2b (aquamarine;eLife 2019;8:e48907), however with significant differences. As before the fibrils are connected by a strong salt bridge between LYS45 and GLU46 (brown arrow), however the pH change from 7.4 to 5.8 causes probably a change of a protonation state of HIS50 (blue arrow) and protonated histidine induces larger structural changes that are absent in the published polymorph 2b. Mechanistic understanding of such structural changes is relevant for studying Parkinson`s Disease.
Manuscript is under preparation for the collaborative project with Riek Group, ETH Zurich. The cryo-EM data was collected on Titan Krios using the K3 detector at ScopeM. Furthermore, for data processing pipelines established at ScopeM in collaboration with the Euler cluster, ETH Zurich team were used.
Main contact: Dr. Bilal Qureshi (email) |
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| Combining techniques across scales to localize atoms in printed nano-structures |
This contribution demonstrates the multifaceted & diverse characterization facility that is ScopeM. We present metallic nano-printed pillars that were made by a cutting-edge automated technique developed in D-MATL’s Laboratory for Nanometallurgy group called electrohydrodynamic redox printing. PhD candidate Maxence Menétrey first imaged the pillars with 465 nm wavelength photons (in Argon gas) in Prof. Spolenak’s lab during the printing process (fig.A, nozzle to pillar distance 7.5 µm), followed by characterizing them applying multiple electron and ion beams at increasing magnifications. An electron beam accelerated to 3kV (in high vacuum) was applied in a Scanning Electron Microscope (fig.B), followed by further interrogation & preparation using a 30kV Focused Ion Beam (fig.C) and imaged with 200kV electrons in a Transmission Electron Microscope (fig.D). Finally, the elemental makeup of the pillars were characterized in 3-dimensions by field ionization (in ultra high vacuum) with Atom Probe Tomography (fig.E: dimensions in nm, each dot representing atomic positions and yellow surfaces highlighting regions of >2 at.% H). This combination of techniques - spanning over 6 orders of magnitude in spatial resolution - in a correlative fashion, enabled the quantification of modulations in microstructures – simply due to a difference in 6V during printing – corresponding strength (mechanical properties), & the 3-dimensional chemical distributions (to < 0.1at.% accuracy, fig.F) within the sub-micrometer printed pillars.
Main contact: Dr. Stephan Gerstl (email) |
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References:
Reiser, A. et al. Multi-metal electrohydrodynamic redox 3D printing at the submicron scale. Nat. Commun. 10, 1853 (2019).https://doi.org/10.1038/s41467-019-09827-1 |
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| ZIDAS 2022: training in Image Analysis for Life Scientists |
| Switzerland Image and Data Analysis School (ZIDAS) is a focused one-week school providing hands-on introductions to image processing and analysis, with priority given to biologically relevant examples. Participants learn the fundamentals of image analysis, including basic macro programming in ImageJ/Fiji as well as other software solutions. By the end of the course, all students have a practical working knowledge of how to attack image analysis problems. ScopeM is a founder organization responsible for the school. In 2022 the school will take place for the sixth time this summer. To be notified when the time and place has been fixed and application is possible, visit the website and sign up with your email address: www.zidas.org |
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| Deep Learning for Image Analysis offered as a service in ScopeM |
| Neural Networks-based methods are nowadays powerful tools for microscopy and can outperform conventional image pipelines opening new possibilities. Some applications of Deep Learning include denoising, increasing resolution (spatial and temporal), reducing the number of markers, segmentation, and detection. Still, accessing these powerful methods is not easy, and often there exists a barrier that novice users find difficult to overcome. The Image and Data Analysis unit of ScopeM offers consultations and support for projects involving Image Analysis, with a special focus on Deep Learning. For more information please check the Image Clinics section on ScopeM’s webpage. |
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