Invited Tutorial

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:

Peng Zeng  | ETH Zürich | Switzerland

https://scopem.ethz.ch/education/MTP.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.

 

Invited Speakers

Patrick Cleeve | Monash University | Australia

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

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.

stay tuned for further updates!