Arctis WebUI – A Novel Software Concept for Automating Cryo-lamellae Production

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. Key components of Arctis PFIB. Novel, small volume vacuum chamber with cryo-box to keep sample contamination levels at a minimum, SEM column for electron imaging, PFIB column featuring three ion species (Xe, Ar, O) for milling, integrated fluorescence microscope (iFLM) for optical targeting, GIS and ion beam sputter target for deposition of protective and conductive layers, CompuStage allowing extended tilt of over 180 degrees, and Autoloader for storing and automatic loading of 12 sample grids.. The cooling system allows for autonomous periods of operation similar to what is possible on Autoloader -TEMs. Screenshots fromArctis WebUI software guiding the user through the complete workflow for cryo-lamellae preparation. (a) Project overview with corresponding grid maps of the loaded samples. (b) Ion image at milling angle with applied milling patterns. (c) Correlative overlay and verification of fluorescence on the final polished lamella.