Microscopy Conference 2023

Session chairs

Focused ion beam (FIB) technique combined with scanning electron microscopy (SEM) enabling nm-precise sample preparation for a broad range of applications in semiconductor industry, material- and life science.  One major application is the preparation of cross sectional and planar samples for electron microscopy analysis. Recent developments of inducted plasma ion sources and combined laser ablation provide increased throughput over conventional Ga-FIB for large area milling.   
 In life sciences, the use of combined  FIB / SEM systems opened new applications for researchers to analyse cellular and multi-cellular systems by slice and view volume imaging and to produce thin cellular lamellae suitable for high-resolution in situ transmission electron tomography. The further development of correlative light and electron microscopy approaches are now opening new avenues for the precise targeting and imaging of unperturbed native environments.
This session welcomes contributions from recent advances in sample preparation and the development and implementation of new hardware and software solutions for FIB milling approaches in material and life sciences, including automated lamella preparation, volume imaging, correlative workflows, and lift out approaches both at room and cryogenic temperature.


Mesoscale sample preparation by multi-Ion-species-plasma-FIB

Abstract text (incl. figure legends and references) Sample preparation for synchrotron X-ray tomography and spectroscopy experiments often requires a target sample size on the order of several tens to hundreds of micrometers. Focused Ion Beam (FIB) based techniques are the most versatile solution: Delicate or brittle materials that are hard to machine mechanically can be dealt with and site-specific preparation is straight forward. Generally Ga-FIB tools can be employed, however, the milling times may be quite long. Here Plasma-FIB instruments offering up to few microamperes of ion current can offer time savings, but also enable to reach dimensions that are simply not manageable by Ga-FIB. In this talk several sample preparation tasks and their solution will be discussed. The materials range from rock to 3D printed alumina over to embedded polymer electrolyte fuel cells and solid state lithium ion batteries. Different sample geometries (windows, pillars and thick lamellae) are covered, challenges and solutions for in-situ lift-out and mounting are discussed. Fig. 1. SEM image of 4 uA Ar-PFIB window with an artificial "crack" milled into 3D printed alumina. Sample courtesy of Fabrizio Verga, Inspire /Wegener group, ETH Zürich.

Workshop 2: Tricks and pitfalls for FIB sample preparation in materials and life sciences

Invited talk

Faster mm-scale defect/failure analysis by combining plasma FIB milling and laser ablation

Abstract text (incl. figure legends and references) The success in materials research is to a certain extent determined by the characterization efficiency. Through faster analysis of materials properties, the performance of engineering materials is improved, better products in the semiconductors and microelectronics industry are developed; and advanced nanomaterials optimize properties of catalysts, coatings, polymers, and many more. It is obvious, that developing new products and technology is very challenging. One area where laser ablation along with FIB-SEMs plays an important role is Failure Analysis (FA). The goal of FA is to determine the defect&quot;s root cause and provides feedback to maintain feasible time-to-market and product reliability. This must be done as precisely as possible and as fast as possible. Industry-proven plasma FIB technology serving these purposes for nearly a decade. Recently, however, significant increases in both the complexity of the devices and the depth of buried defects, together with pressure on time-to-result may push the plasma FIB speed to its limit. There are different methods how to further enhance sample preparation throughput of a Plasma FIB-SEM without sacrificing the quality of the data—such as the utilization of rocking polishing or mask method—nevertheless, ultimate throughput can be achieved by the smart implementation of the laser ablation technique in some steps of the Failure Analysis workflow. Through the purpose-designed workflow, the plasma FIB sample analysis efficiency is increased, time to the results decreased while new synergies among laser ablation and FIB-SEMs are opened. In this talk, you will learn about TESCAN Large Volume Workflow. We will guide you through the use cases where wide range of materials and complex devices were investigated for their failures. By the end of the talk, we disclose the method which is used to achieve fast, artifact-free sample preparation while achieving high-quality results proven by UHR SEM imaging.

Prof. Dr.

Next generation sample preparation for cryo-ET – instruments, accessories and workflows

Abstract text (incl. figure legends and references) The recent technological breakthrough in cryo-electron tomography (cryo-ET) opens the possibility of imaging macromolecular complexes in their native cellular context at sub-5Å resolution [1,2]. This technology is the foundation for an emerging field in the life sciences, often termed structural cell biology, capable of revealing how different conformational states of protein complexes are linked to biological functions. However, imaging is highly dependent on sample preparation. This is especially true for transmission electron microscopy (TEM), where samples must be thin enough to provide high-resolution information, homogeneous in thickness for consistent high-quality data, and in their original state, as in cryo-TEM, to preserve the structural features of macromolecules. Sample preparation, while largely underappreciated, is thus a vital part of imaging. Since its beginnings as a sample-thinning technique for cryoET, cryogenic focused ion beam milling (cryoFIB) using gallium ions has evolved into a procedure that encompasses several different methods. Methods like on-the-grid lamella-preparation of plunge frozen samples, iterative FIB ablation and scanning electron microscopy (SEM) imaging, and most recently lift-out for high-pressure frozen (HPF) multicellular samples [3,4]. The latter is an established method in materials science, but since its introduction for cryo-ET, it has been used rather rarely [5]. The reasons why the lift-out method falls short compared to lamellar preparation on the grid are quite obvious: First, it is very time consuming to mill high-pressure frozen (HPF) samples that are up to 200 microns thick; second, the whole process is often disappointingly inefficient, mainly because of its complexity; third, there is no way to assess ice quality before or during preparation, only after the first TEM image does one know whether the ice is "good" or "bad"; and finally, the "survival rate" of lift-out lamellae is low, either they are lost during manual transfer or they are contaminated beyond recognition. Yet, there must be ways to streamline this process and make multicellular specimens and small organisms amenable to routine access in a reasonable time frame. PlasmaFIB technology promises to do just that: Higher beam currents and higher ablation rates to make bulky samples easily accessible, hardware automation that frees the user from any manual operation and increases throughput and survival rates, and finally integrated solutions for targeting (correlative fluorescence microscopy, CLEM) and ice quality assessment. Here we present the latest developments towards full automation for plunge-frozen and high pressure frozen samples, as well as initial results and experiences with a novel plasma cryo-FIB system specifically designed for large scale cryo-FIB milling. References: [1] D. Tegunov et al., Nature Methods 18, 186–193 (2021). [2] S. Khavnekar et al. bioRxiv; DOI: 10.1101/2022.06.16.496417 (2022). [3] S. Klumpe et al., eLife 2021;10:e70506 (2021). [4] S. Klumpe et al., Microscopy Today 30(1), 42-47 (2022). [5] M. Schaffer et al., Nature Methods 16, 757-762 (2019).

LS 2: Cryo-tomography

State of the art of volume imaging and CLEM of organoids

Abstract text (incl. figure legends and references) Organoids recapitulate tissue architectures and represent an opportunity to investigate healthy or diseased tissues. Understanding organoid complexity requires the integration of diverse imaging modalities covering different spatial and temporal resolutions. Fluorescence light microscopy allows studying the cellular sociology in living samples. FIB-SEM volume imaging can further probe the multicellular environment at ultrastructural resolution. Cryo-electron tomography (cryoET) bridges the gap with atomic resolution to investigate macromolecular complexes in the cell. Further, deep-learning software is required to correlate and quantitatively analyse these data. Here, we present the state of the art and future direction of light and electron microscopy imaging of organoids both at room temperature and cryogenic regime. Matrigel-embedded organoids were either cultured directly in HPF carriers1 or multi-well dishes using liquid medium. These carries were used to perform confocal live-cell imaging using cell-permeable fluorescence dyes. After high-pressure freezing, the fluorescence signal guided FIB-SEM volume imaging acquisitions at low temperature. Further, sample thinning for cryo-electron tomography was achieved by lift-out. Alternatively, organoids in HPF carriers were processed by freeze-substitution and resin embedding follow by laser branding to FIB-SEM volume imaging acquisitions2. FIB-SEM was done on a performed on a Zeiss CrossBeam XB550 and on Thermo Fisher Scientific Aquilos. Lastly, correlation of light and electron microscopy data as well as automatic image segmentation was done by ORS Dragonfly. We show how to make organoid delicate specimens amenable to high-pressure freezing and fluorescence light microscopy both a room temperature and cryogenic regime. We compare the advantages of live dyes versus genetically encoded fluorescent reports and we show the limits of fluorescence light microscopy in imaging large (&gt; 200µm) and deep (&gt; 20µm) biological samples. Then, we illustrate correlative light and electron microscopy approaches for organoid samples. Further, we describe our results of FIB-SEM volume imaging at ultrastructural resolution of organoids at room temperature and the current limits of imaging at low-temperature. To provide a quantitative understanding of the complex and heterogenous multicellular environment of organoids, large FIB-SEM dataset need to be annotated. While manual annotation becomes unfeasible given the large amount of data, deep-learning algorithms offer a workable solution for automating this task. We used trainable convolutional neural networks provided in the software ORS Dragonfly for automatic segmentation of subcellular structures. We focused our attention especially on the interaction between cortical cytoskeleton and cell junctions in the context of patient-derived cancer organoids. Finally, we provide an overview of the actual possibilities to perform lift-out from high-pressure frozen samples for cryoET imaging. Ronchi, P. et al. High-precision targeting workflow for volume electron microscopy. J. Cell Biol. 220, (2021). D&quot;Imprima, E. et al. Integrated light and electron microscopy continuum resolution imaging of 3D cell cultures. bioRxiv 2021.07.02.450855 (2021) doi:10.1101/2021.07.02.450855.

WS 2
Tricks and pitfalls for FIB sample preparation in materials and life sciences

Appointment

Session chairs

Description

Programme

WS 2Tricks and pitfalls for FIB sample preparation in materials and life sciences

Appointment

Session chairs

Description

Programme

WS 2
Tricks and pitfalls for FIB sample preparation in materials and life sciences