Cryogenic-electron tomography (cryo-ET) permits the in situ visualization of biological macromolecules at the molecular level. Owing to the variable thickness of cells, tissues and organisms, frozen specimens may need to be thinned by cryo-focused ion beam (FIB) milling to produce thin (<500 nm) cryo-lamellae suitable for cryo-ET. Locating regions of interest remains a challenge because untargeted milling can lead to inadvertent ablation and removal of regions of interest. Correlative light and electron microscopy, combined with cryo-FIB milling, can guide the identification of labeled targets in the cellular milieu. Multiple transfers between cryo-imaging instruments, cumbersome correlation algorithms, limited accuracy and low throughput have hindered the routine adoption of cryo-FIB milling within a multimodal correlative workflow for in situ structural biology. Here we present a workflow for 3D correlative cryo-fluorescence light microscopy-FIB-ET that streamlines fluorescence light microscopy-guided FIB milling, improving throughput while preserving both structural and contextual information. The complete integration of hardware and software described here minimizes sample contamination from cross-platform exchanges and greatly enhances the efficiency of 3D targeting in cryo-milling. We then describe procedures for implementing montage parallel array cryo-ET (MPACT), which can be easily adapted to any modern life-science transmission electron microscope. MPACT supports high-throughput cryo-ET acquisitions (10 tilt series in 1.5 h) for structure determination and comprehensive contextual understanding of macromolecules within their native surroundings. A complete session from sample preparation to MPACT data processing takes 5−7 d for an individual experienced in both cryo-EM and cryo-FIB milling. A protocol for cryogenic 3D correlative focused ion beam milling using an integrated fluorescence light microscope and montage cryo-ET for nonadherent and adherent mammalian cells, as well as primary Drosophila melanogaster neurons.

Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.

Journal Article Precise 3D Localization by Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET Get access Jae Yang, Jae Yang Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, United States Search for other works by this author on: Oxford Academic Google Scholar Veronika Vrbovská, Veronika Vrbovská Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Tilman Franke, Tilman Franke Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Bryan Sibert, Bryan Sibert Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesCryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, United States Search for other works by this author on: Oxford Academic Google Scholar Matt Larson, Matt Larson Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesCryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, United States Search for other works by this author on: Oxford Academic Google Scholar Tom Coomes, Tom Coomes Thermo Fisher Scientific, Hillsboro, OR, United States Search for other works by this author on: Oxford Academic Google Scholar Alexander Rigort, Alexander Rigort Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar John Mitchels, John Mitchels Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Elizabeth R Wright Elizabeth R Wright Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesCryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesMidwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United States Corresponding author: erwright2@wisc.edu Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1055–1057, https://doi.org/10.1093/micmic/ozad067.541 Published: 22 July 2023

Cryo-electron tomography (cryo-ET) is a revolutionary and powerful method for in situ structural biology.This method provides high resolution insights into cells, their organelles, proteins and macromolecular complexes, and the structural organization of the inner cellular space.In combination with image processing by subtomogram averaging (STA), cryo-ET allows sub-nanometer resolution structure determination of biological macromolecules.The Arctis cryo-Plasma Focused Ion Beam (cryo-PFIB) is designed for automated, production of cryo-lamellae from vitrified cells.Its robotic sample loading system (Autoloader) provides a unique, direct connection between the cryo-PFIB microscope and the cryo-transmission electron microscope (cryo-TEM).An integrated fluorescent light microscope (iFLM) at the coincidence point of ion and electron beam, enables end-pointing of fluorescently labeled targets inside frozen cells and allows for fluorescence guided lamella preparation.We developed a novel docker based server architecture which enables support for a web based user interface ('Arctis WebUI') covering the complete cryo-lamellae preparation process with a high focus on ease of use and automation.The new WebUI concept combines multi user projects management, customizable experimental user dashboards, sample navigation, feature targeting, and lamella milling into one single dedicated user interface which can be run remotely from anywhere .Due to the autonomous cooling system and the integrated Autoloader, multiple grids can be processed within one project, keeping the microscope in use for several days.Up to now, automated lamella preparation required the use of multiple different software programs for mapping, correlation, targeting and milling.Arctis WebUI aims to streamline the lamella production process, by integrating all these functionalities into a single software package, which includes a series of manual and automated steps.These steps can be customized via templates for different types of samples.The novel server architecture features an intelligent job queuing system enabling maximum microscope utilization.In this contribution we will discuss how the Arctis microscope, together with the newly developed Arctis WebUI software, provides a next step in connectivity to the cryo-TEM and for streamlining the automated production of high quality cryo-lamellae for tomography.

Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for in situ cryo-ET. However, correlative workflows that incorporate precise targeting remain challenging. Here, we demonstrate the development and use of an integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to enable a coordinate-based two-point 3D correlative workflow. The iFLM guided targeting of regions of interest coupled with an automated milling process of the cryo-FIB-SEM instrument allows for the efficient preparation of 9-12 ∼200 nm thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high pressure frozen tissue, to produce lamellae for downstream cryo-ET data collection.

Journal Article Fluorescence-guided Cryo-lift-out using an Integrated Fluorescence Light Microscope and an Optimized Sample-needle Attachment Procedure Get access Veronika Vrbovská, Veronika Vrbovská Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Sven Klumpe, Sven Klumpe Research Group CryoEM Technology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for other works by this author on: Oxford Academic Google Scholar Christopher Thompson, Christopher Thompson Thermo Fisher Scientific, Eindhoven, Netherlands Search for other works by this author on: Oxford Academic Google Scholar Alexander Rigort, Alexander Rigort Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar John Mitchels, John Mitchels Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Tilman Franke, Tilman Franke Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Michaela Müllerová, Michaela Müllerová Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Anna Kasáková, Anna Kasáková Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Miloš Hovorka Miloš Hovorka Thermo Fisher Scientific, Brno, Czech Republic Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 29, Issue Supplement_1, 1 August 2023, Pages 1051–1052, https://doi.org/10.1093/micmic/ozad067.538 Published: 22 July 2023

Dual-beam cryo-FIB/SEM microscopes have become increasingly indispensable tools for cryo-electron tomography over the past decade. Thermo Fisher Scientific has played a leading role in the development together with pioneering scientific groups in the field, setting standards for this still young class of cryo-dedicated instruments. Microscopes such as the Aquilos 2 are now in their second generation and, with new automation routines, along with integrated cryo-lift-out and fluorescence microscopy options, have steadily expanded the application range of the cryo-FIB and are already routinely used in many laboratories for cryo-lamella preparation. the advent of cryo-FIB instrumentation, as well as improved cryo-TEM data collection schemes utilizing faster direct electron detectors and modern energy filters, completely new possibilities have opened for cryo-electron tomography as evidenced by the steady increase in the number of publications in which new cell and structural biology questions are elucidated using this method

Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.

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We would like to introduce the new FEI Scios Cryo; a system which is built upon a successful prototype and other variants which have resulted already in significant publications [1,2,3]. This instrument offers customers a dedicated and reliable entry point to the world of ‘in situ’ lamella preparation for cryo electron tomography (cryoET). Cryo-focused ion beam (cryo-FIB)-based sample preparation has recently been shown to extend structural biology from solely purified structures, to the dynamic complexity of the cell [4].

We are presenting a new extension to our Cell and Tissue/Neurobiology large volume imaging workflow, with the goal of increasing acquisition speed by more than five times.Instead of scanning dense square-grid frames, in the conventional way, our approach is here to explore the use of sparse scanning and inpainting techniques inspired by Compressive Sensing (CS) [1].Sparse samples are obtained by pseudo-random scan patterns, and reconstruction algorithms are used to recover the dense volume data.The goal is to recover 3D datasets with minimum loss of information.Techniques inspired by CS gained wide attention over the last decade and are now being used in various applications where sensor bandwidth is a limiting factor.They have been recently explored for SEM and STEM applications [2][3].In the context of nano-scale cell biology volume acquisition, we expect these techniques to ultimately increase the imaging throughput by nearly an order of magnitude.We will discuss additional advantages of this approach, such as the low-dose imaging of sensitive specimens, and the good compatibility with backscatter electron imaging.A key enabler of any sparse scan application to EM is the accurate control of scan locations.It has been shown in [2] and in our own experiments that precise positioning of the beam at the planned sampling locations is essential for a good CS reconstruction.We have developed advanced minimum-path scanning strategies to address this issue.The scanning technique is illustrated in Fig. 1, where the left two images show a conventional raster scan at 300ns dwell visiting a random set of points with the compressive sensing reconstruction obtained from such scan strategy.The right two images of Fig. 1 show an example minimum-path scan pattern and a much improved reconstruction result from images acquired with this second method.In future work we will compare pseudo-random sparse sampling, in combination with a reconstruction algorithm based on CS-inspired in-painting, to conventional grid sampling of the same effective dose, in combination with a de-noising algorithm, also based on CS.CS machine learning algorithms build patch-dictionaries, which are used as the building blocks for data representation [3].During live acquisition runs, such dictionaries can be used to in-paint with high fidelity, the sparsely sampled datasets (Figure 2).We are implementing the new sparse scanning modules on SEM platforms, which also employ the Multi Energy Deconvolution SEM (MED-SEM) technology and Serial Block Face (SBF) imaging [4].By incorporating CS, we will have an instrument allowing for both high-resolution isotropic imaging, and the fast acquisition of very large datasets (Figure 3).

Cryo Electron Tomography is emerging as a technique which enables the extension of structural biology from solely purified structures, to the dynamic complexity of the cell [1].Focused Ion Beam (FIB-SEM) erosion under cryogenic temperatures offers researchers a way of producing damage and artefact free lamella which can be utilized in high end tomography tools such as the FEI Titan Krios™.Several Cryo-FIB prototype tools exist and have been producing significant results utilizing the 'in situ' approach [2,3,4].Correct preparation of samples for in situ lamella prep is critical for making use of this emerging technique.There are many steps where the sample can be significantly contaminated or even completely damaged which would prevent from collection of high-quality cryo-electron tomography data.Standardization of the whole process of ablation of cellular material and sample handling is required to provide sufficient yield of high-quality samples for TEM imaging.Prior to these steps, the protocols for adhesion of the cells on the TEM grids and its subsequent vitrification require standardization.Here, we provide a protocol for preparation of samples for cryofocused ion beam micromachining (FIB-milling) which in our hands provides high reproducibility and quality of the input material for FIB-milling.An example of standardization based on Saccharomyces cerevisiae and A9 are presented, showing the critical steps and success indicators required to reproduce high quality samples.It is hoped that these insights will help others to quickly adapt to this new method of sample preparation.