Workshop information
Below you can find an overview of the workshops you can follow during CLEM2024. In total there are 6 workshop slots: A,B,C,D,E and F. (A,B,C will take place on 20 March, D,E,F on 21 March). This means you can follow 6 out of 10 workshops.
Closer to the event you will receive an email with a submission form. In this form you can to rank the 10 workshops from 1 to 10 based on your own interest. This ranking will be used to assign the workshop slots to each one of you.
When you arrive on the 20th of March at the Technology park you will receive the entrance tickets for all the workshops your are assigned to.
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Workshop 1: X-ray targeted volume EM From Versa and Ultramicrotome (Crosshair) to Volutome
Workshop owners: Christel Genoud - UNIL, CH & Paolo Ronchi, EMBL
This workshop is designed to showcase the application of X-ray imaging in conjunction with serial block-face scanning electron microscopy (SBF-SEM) for targeted the acquisition of a large 3D dataset in a piece of tissue. We will show the power of X-ray tomography to visualize anatomical features of the EM prepared samples within resin blocks. These heavily stained samples are opaque to light and difficult to orientate. The X-ray tomogram provides a 3D detailed map, allowing precise measurements. We will demonstrate the use of the 'crosshair workflow,' as presented by Meechan et al., 2022 (https://doi.org/10.7554/eLife.80899). This makes use of the X-ray dataset to optimize the trimming strategy at the ultramicrotome, exposing a specific region of interest within the sample and therefore reducing the imaging time at the SBF-SEM. Finally, we will illustrate the process of imaging the prepared specimen using a Zeiss Volutome. We will demonstrate how the sample is placed in the microtome and the approach to the diamond knife. We will then show the different microscope parameters and imaging strategies available with the Volutome, going from large tiling dataset possibilities to optimized resolution acquisitions. We will further discuss how to handle the datasets during and after image acquisition.
Workshop 2: Femtosecond laser milling of biological tissues for nanoscale imaging
Workshop owners: Carles Bosch & Christopher Peddie (The Francis Crick Institute, UK)
Biological tissues contain multicellular functionally relevant systems. Reaching a mechanistic understanding of those systems is challenged by their inherently multiscale and multimodal nature. Structures of interest might be subcellular features a few nm wide, such as cell membranes, and may extend to volumes >1 mm3 containing entire functional units, such as neuronal circuits. Orthogonal information provided by multimodal analysis also brings crucial insights. Correlative multimodal imaging (CMI) pipelines break down these challenges experimentally, linking in vivo and ex vivo insights from light, X-ray, and volume electron microscopy. A crucial step in CMI pipelines is targeted trimming of specimens into isolated volumes compatible with downstream high-resolution imaging technologies. Femtosecond lasers can rapidly mill arbitrary shapes in a wide range of materials. The application of fsLaser milling as a preparation tool for resin-embedded specimens has many advantages, including optimisation of sample geometry for specific imaging techniques, and the extraction of multiple samples at targeted locations. In this workshop we explore the use of fsLaser milling to generate sample arrays from a parent ~4.5 mm3 volume of mouse brain by analysing pre-milling X-ray CT scans, sharing insights on the milling process, by manipulating pillars, and by imaging an extracted pillar with FIB-SEM.
Workshop 3: Correlative Array Tomography
Workshop owners: Jemima Burden (UCL, UK) & Ian White (UCL, UK)
SEM Array tomography is a non-destructive volume EM technique that allows considerable flexibility in sample preparation which can include correlative options. Ultrathin sections of a sample are collected onto either, a solid support (e.g. coverslips using an ultramicrotome) or onto tape (using an ATUMtome) before EM imaging. In correlative workflows, light microscopy can be performed prior to EM embedding, after embedding or after sectioning as appropriate, and these images can be imported into the Atlas SEM acquisition software to locate areas of interest and enable easy targeting of automated, high resolution ultrastructural imaging in the SEM. This workshop will cover many aspects of this workflow, with several of the key techniques being demonstrated.
Workshop 4: Power hour with molecular tools and spatial analysis experts
Workshop owners: Anneke Kremer (VIB,BE), Evelien Van Hamme (VIB,BE), Ben Giepmans (UMC, NL)
It is all about the whereabouts. Identifying and locating a specific molecule, structure or cell within a 3D EM dataset is often compared to finding a needle in a haystack. However, if we are guided by fluorescence, a light microscope can reveal the approximate whereabouts, including following dynamic observations in a living biosystem prior to 3D vEM sample preparation and acquistion.
For this power hour, we’ll delve into the diverse spectrum of markers that can be used for CLEM workflows (‘the CLEM toolbox’) – whether fluorescent, peroxidase-based or gold-labeled; whether structure, protein and/or RNA markers. In addition, we will highlight the upcoming possibilities of using the endogenous information of the biosample for analysis, like RAMAN techniques at the LM level as well as elemental analysis at the EM level. We invite you to bring your challenging specific projects and inquiries for an interactive discussion aimed at offering guidance and insights tailored to your needs and challenge us to make the invisible visible!
Workshop 5: Alignment, visualization and sharing of correlative data
Workshop owners: Julian Hennies (EMBL, DE), Benjamin Pavie (VIB, BE) & Tatiana Woller (VIB-KU Leuven, BE)
Within this workshop will teach the basics of how to navigate large 3D datasets using the MoBIE browser, including how to set up a MoBIE project with data of correlative light and electron microscopy experiments. Additionally, we will briefly show project generation using the MoBIE python API which enables automated MoBIE project creation specifically for your custom project. In a second part, we will show how to publish data on image repositories such as BioImage Archive and EMPIAR, and how it could be fully integrated into an institute data repository.
It's necessary to bring your own laptop for this workshop.
Workshop 6: Power hour on AI tools and public data archive
Workshop owners: Kedar Narayan (NIH, US) & Ilya Belevich (University of Helsinki, FI)
Segmenting features from volume EM datasets is a known problem in experimental pipelines, and recent advances that have increased throughput have further magnified this challenge. In connectomics research, segmentation tools have moved the field forward by helping to create "wiring diagrams" at scale from neuronal specimens. However, for questions in the wider cell biology research space, segmenting features of interest such as organelles - which tend to be heterogenous and numerous - continues to be problematic. Computer-aided and manual segmentation have helped greatly, yet cannot keep up with scale. Following recent and dramatic innovations in this space using AI, advances in deep learning approaches are now addressing this problem and computational solutions usable by biologists are emerging. In this tutorial for beginners, Ilya Belevich and Kedar Narayan will lead the group through a sampling of the current landscape of artificial intelligence as it pertains to EM segmentation. Participants will be introduced to some basic concepts in deep learning, and will be guided through several open-source plugins and tools such as MIB, SAM and empanada that will help tackle segmentation problems. At the end of the session, participants will learn to deploy some of these tools and will gain the confidence of using, extending and tailoring these powerful approaches to their own research needs.
It's necessary to bring your own laptop for this workshop.
Workshop 6 - Software guidelines
Workshop 7: Power hour on different light microscopes for correlative microscopy
Workshop owners: Eef Parthoens (VIB, BE), Nikky Corthout (VIB,BE), Amanda Goncalves (VIB-U Gent, BE) & Jeremy Verbeke (VIB-U Gent, BE)
Join our workshop on Correlative Light and Electron Microscopy (CLEM) with a primary focus on the light microscopy component. This concise and dynamic session will provide a comprehensive overview of three key techniques: laser branding, gridded coverslips, and fiducials markers. The workshop will start with a brief introduction on the importance in bridging the gap between light and electron microscopy. Laser branding will be explored, offering insights in how to successfully integrate in an experiment workflow. The module dedicated to cells on gridded will explore streamlined workflows for efficient CLEM, emphasizing optimized light microscopy settings. This workshop will conclude with a detailed examination of fiducial markers, elucidating their pivotal role in achieving accurate correlations between the two microscopy modalities. Participants can anticipate a hands-on experience, practical demonstrations, and engaging discussions with experts in the field. This workshop will provide attendees with practical knowledge to enhance their CLEM methodologies, specifically in the realm of light microscopy.
Workshop 8: Starting CLEM in a core facility setting
Workshop owners: Saskia Lippens (VIB, BE) & Paul Verkade (University of Bristol, UK)
Microscopy plays a crucial role in nearly every life science research endeavor, with an increasing demand for Correlative Light Electron Microscopy (CLEM) approaches across diverse fields. The proliferation of microscopy cores since the end of the last century has positioned these facilities as vital hubs for researchers seeking advanced CLEM workflows. However, the multitude of approaches presents a challenge in establishing effective setups. The necessity to customize workflows for specific biological questions may conflict with the overarching goal of fostering robustness, simplicity, and throughput within a facility. This workshop endeavors to explore several aspects of implementing and developing complex protocols, procedures, and technologies associated with CLEM. We’ll discuss the delicate balance for adaptability to diverse biological inquiries with the facility's overarching principles of robustness and efficiency. Furthermore, the workshop will explore the convergence of two technology fields of light microscopy and electron microscopy in order to perform CLEM and the critical roles different experts and scientists play in executing such projects within a core setting. By addressing the challenges and opportunities inherent in merging these fields, we hope to foster a comprehensive understanding of how best to navigate the integration of diverse imaging technologies for life science research. Your insights and experiences are highly valued, and we look forward to engaging in thoughtful discussions during the workshop.
Workshop 9: High-throughput imaging with ZEISS Multi SEM
Workshop owners: Anna-Lena Eberle, ZEISS, DE & Helmstaedter Lab (MPI for Brain Research, DE)
Over the past several years, scanning electron microscopy has become a standard method for 3D tissue volume-imaging experiments. However, all scanning technologies are inherently slow, so for large-scale experiments a faster acquisition workflow is needed. The multibeam SEM from ZEISS utilizes up to 91 electron beams in parallel to scan the sample. Of the various approaches existing for the acquisition of 3D tissue data, a workflow in which ultra-thin serial sections are obtained and then imaged sequentially is the most established one for the multibeam SEM. Here, we want to give an overview of the high-throughput serial section acquisition workflow with the ZEISS MultiSEM. (Image reference: DOI: 10.1126/science.abo0924)
- Part of this workshop will be presented remotely -
Workshop 10: In Resin Fluorescence Preservation
Workshop owners: Katlijn Vints (VIB-KU Leuven, BE), Dumisile Lumkwana (The Francis Crick Institute, UK) & Malgorzata Sliwinska (VIB KU Leuven, BE)
For decades, Correlative Light and Electron Microscopy (CLEM) has been of great value to locate fluorescently labelled targets imaged with a light microscope (LM) within the ultrastructural context observed by an electron microscope (EM). The most accurate correlation is obtained when the colocalisation of a fluorescent signal with the ultrastructural details is done in the same 2D plane. A well-known approach to achieve this is the In-Resin Fluorescence (IRF) technique, where fluorescence is preserved in ultrathin resin sections of samples embedded for EM, allowing for sequential imaging of the same position using two modalities: LM and EM. The Bio Imaging Core of VIB in Belgium will demonstrate a standard successfully working protocol for maintaining fluorophores in a wide variety of resin embedded samples using a High-Pressure Freezer. The Francis Crick Institute will demonstrate a new protocol that does not require a high-pressure freezer to produce IRF blocks, developed in collaboration with Imperial College and the Instituto Gulbenkian de Ciência. This protocol is part of the newly developed Visual Proteomics CLEM-KIT, a cost-effective pipeline for high resolution volume CLEM that can be implemented in any light microscopy facility by removing the dependency on advanced EM expertise and equipment.
