Invited Tutorials
Carsten Putzke | MPI Hamburg | Germany
https://www.mpsd.mpg.de/person/114295/633655
Automation in Focused Ion Beam and Scanning Electron Microscopy
Automation of repetitive tasks is a common challenge of many fields in achieving higher productivity and reliability. The particular challenge in fundamental science is the vast variety of different materials and tasks. The combination of both presents an obstacle that requires higher efforts in programming of automated tools. The benefit lies in an enhanced accuracy and the possibility for the user to focus on progress rather than repetition.
In this tutorial we will look into:
- The tools currently available to automation in FIB/SEM.
- The basic requirements for a successful automation and how automation enables automation.
- Obstacles of automation of FIB/SEM in material science.
- Examples of FIB/SEM automation in alignment and processing.
- Live programming and execution of an example in Python.
Peng Zeng | ETH Zürich | Switzerland
https://scopem.ethz.ch/center/people/person-detail.MjU1MjE5.TGlzdC81MzIsOTkxMzExMjcy.html
FIB automation: TEM lamellae for High Resolution TEM/STEM
FIB is widely used to prepare TEM lamellae and due to its ability to mill almost all materials, it is a powerful tool for all materials. In addition, FIB resolution allow us to locate the lamellae position precisely (in the range of nm), make it a unique tool for site-specific TEM lamellae preparation. Compared with traditional methods (cutting, polishing and ion milling/electropolishing), FIB is also an efficient tool. With the FIB automation, now we could reliably produce high quality TEM lamellae under 1-1.5 hours.
In this tutorial, we will present our experiences of TEM lamellae preparation using FIB automation and discuss the best approach for the TEM lamellae preparation for high resolution TEM/STEM.
Zineb Saghi and Thomas David | CEA Leti | France
Tomographic data processing using machine learning and deep learning methods
Extracting reliable information from 3D tomographic data is a complex task that involves challenging steps such as artifact detection and correction, denoising and segmentation. In this tutorial, we will illustrate the potential of machine learning and deep learning methods to perform these tasks with minimal human intervention. Using a remote server, we will make two short demos:
(1) Semantic image segmentation using Ilastik toolkit;
(2) Correction of missing wedge artifacts in limited-angle tomography, using U-net.
Mahdi Ghorbani-Asl | HZDR Dresden | Germany
Simulation of irradiation effects in solids
The behavior of the materials under the influence of energetic ions has been extensively studied through theoretical simulations.* These techniques can provide details on ion ranges, energy losses, as well as on the types, stabilities, and evolution of irradiation-induced defects. We give a brief introduction to the computational methods that are commonly used to simulate the effects of radiation on both bulk and nanostructured solids. We discuss the fundamental physics that underlies both binary collision approximation (BCA) and more precise molecular dynamics simulation techniques. We use a few examples from recent theoretical and experimental efforts to illustrate the differences between the impacts of ions on three-dimensional and two-dimensional materials. Finally, some short tutorials on the simulation of ion irradiation using SRIM code will be provided.
Invited Talks
Patrick Cleeve | Monash University | Australia
https://www.demarco-lab.com/members
OpenFIBSEM: A universal API for FIBSEM control, development and automation
Automation in microscopy is the key to success in long and complex experiments. Most microscopy manufacturers provide Application Programming Interfaces (API) to enable communication between a user-defined program and the hardware. However, these APIs are manufacturer specific, and require detailed knowledge about the individual systems.
In this talk we present OpenFIBSEM, an universal API for FIBSEM control, development and automation. The API aims to provide a single interface for controlling FIBSEM systems, as well as providing reusable components for microscopy workflows. OpenFIBSEM is currently in development and supports ThermoFisher and Tescan microscopes. We will discuss the API design, development process, and the application model. We will also discuss the applications we are developing using OpenFIBSEM including AutoLamella, AutoLiftout, and more.
The API and applications are open source and available at https://github.com/DeMarcoLab/fibsem.
Gregor L. Weiss | ETH Zürich | Switzerland
https://pilhoferlab.ethz.ch/people/gregor-weiss.html
Combining cryo-FIB milling with cryo-electron tomography enables macromolecular insights into biological samples
Imaging flash-frozen biological samples by cryo-electron tomography (cryoET) has become a powerful method for different research fields in biology. CryoET is a modality of cryo-electron microscopy and resolves macromolecular complexes in their native cellular context and at several nanometers of resolution.
However, cryoET is limited to samples that have a thickness well below 800 nm. For thicker samples, cryo-thinning techniques must be applied to overcome this limitation. While cryo-sectioning could generate some insights, the technique is challenging, cannot be automated, and introduces artifacts that prevent high-resolution imaging approaches. The adaptation of cryo-focused ion beam (FIB) milling to prepare thin electron-transparent lamellae through biological samples enabled a multitude of groundbreaking insights into the structure and function of cells – from bacterial to eukaryotic model organisms. The throughput of cryo-FIB milling for biological samples was very low at the beginning and required a large amount of user input. To tackle this bottleneck, we developed in collaboration with Zeiss a procedure to automate the processes to sequentially mill multiple targets, which significantly increased the throughput. The thinning of well-established model organisms using cryo-FIB milling for subsequent cryoET studies is nowadays routine and is becoming more and more popular. However, the application of this thinning technique to more complex and large samples, such as environmental or clinical samples, remains challenging.
During my talk, I will present our latest efforts in applying a combined imaging workflow to more complex biological samples, with a specific focus on clinical samples such as body fluids or tissue biopsies. In this approach we use cryo-light microscopy to target rare events for subsequent correlated cryo-(plasma) FIB milling for sample thinning, cryo-volume imaging for the integration of imaging datasets across scales and cryoET for high-resolution structural analysis.
Antje Biesemeier | LIST | Luxembourg
High-resolution chemical imaging in the cold: A cryo-FIB platform for correlative topographical and in-depth analysis of frozen-hydrated biological specimens
Antje Biesemeier1 *, Tatjana Taubitz1, Olivier De Castro1, and T. Wirtz1
1 Advanced Instrumentation for Nano-Analytics, Materials Research and Technology Department, Luxembourg Institute of Science and Technology, Belvaux, Luxembourg.
* Corresponding author: antje.biesemeier@list.lu
Biological specimens for high resolution (HR) imaging and chemical analysis usually suffer from harsh sample preparation needed for in vacuum-analysis in the electron microscope (EM). Artefacts related to sample fixation and epoxy-embedding or drying, affect ultrastructure as well as elemental content (redistribution or washout of water-soluble molecules and lipids). Thus, cryo-electron microscopy has become a straightforward technique for topographic imaging and in-depth (i.e. subsurface and bulk) analysis. However, and independently of the temperature, chemical information of e.g. individual nanoparticles as they are studied in nanotoxicological research topics, can often not be resolved within the biological matrix by analytical EM.
As an alternative, here, we present a cryo-FIB (focused ion beam) platform equipped with secondary electron (SE), secondary ion mass spectrometry (SIMS) and scanning transmission helium ion microscopy (STIM) detection capability that can be operated at room temperature (RT) or under cryogenic conditions (<-145°C) [1]. The microscope is based on an ultra-high brightness Gas Field Ion Source (GFIS) allowing to scan the samples with very finely focused He+ and Ne+ ion beams and typically operated in the < 35 keV energy range. Sub-nanometer spatially resolved SE images with a high depth of field and topographic contrast are predominantly obtained with the He+ ion beam. Sub-surface or volume information at nanometre scale can be retrieved from thin (about 100 nm thickness) samples using the recently developed STIM detection system [2] located below the sample. Compositional information, e.g. for identification and subcellular localisation of individual metal nanoparticles embedded in biological matrices can be obtained with the Ne+ beam and using the incorporated compact magnetic sector SIMS system [3]. Its micro channel plate (MCP) – delay line (DL) based continuous focal plane detector features parallel mass detection for each scanned sample pixel over the selected mass range, offering hyperspectral SIMS imaging with high sensitivity and at sub-15 nm lateral resolution. Key for cryo-FIB investigations is the integration of a piezo-driven 5-axis cryo-stage along with cryo-capabilities for sample transfers. In combination with dedicated cryo-sample preparation equipment, e.g. a specialised low humidity nitrogen atmosphere glovebox, the cryo-FIB platform is an ideal tool for in-situ correlative studies not only for biological but also other beam sensitive materials like polymers. Exemplary data will be presented from the field of nanotoxicology, medicine and beyond [4].
[1] O. De Castro et al., Anal. Chem. 2021, 93 (43), 14417–14424.
[2] E. Serralta et al., Beilstein J. Nanotechnology 11 (2020), p. 1854.
[3] J.-N. Audinot et al., Rep. Prog. Phys. 84 (2021) 105901
[4] This work has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 720964 and was supported by the Luxembourg National Research Fund via the projects INTER/DFG/19/13992454 and FNR CORE C21/BM/15754743.
Hilary Noad | MPI Dresden | Germany
https://www.cpfs.mpg.de/person/105429/1454854
Tuning and probing the electron system in a quantum material with the help of FIB processing
A central approach in the study of quantum materials is to use tuning parameters such as magnetic field, chemical composition, or pressure to modify the interactions between electrons in a controlled way. I will discuss two examples of using a focused ion beam (FIB) to achieve new levels of tunability in a quantum material. While the methods that I will present are broadly applicable, I will focus on their application to Sr2RuO4, a material that behaves as a conventional metal at low temperatures and exhibits unconventional superconductivity below 1.5 K. In the first example that I will present, we used a plasma FIB to sculpt samples for stress-strain measurements under uniaxial pressure, enabling us to quantitatively extract the Young’s modulus as a function of strain. These measurements reveal an unexpectedly large softening of the lattice, driven by conduction electrons, at a Fermi surface topological transition. In the second example, we used FIBs to prepare microstructures of Sr2RuO4 that were compatible with repeated high-energy electron irradiation. With these samples, we systematically tuned the superconducting transition temperature by varying the concentration of point defects, confirming the unconventional nature of the superconductivity.
stay tuned for further updates!