Community workshop

Advancements and Applications of Open Hardware in Microscopy (CW1)

CW Organizer:

Gabriel Cristobal

Instituto de Óptica/Spain

Gloria Bueno

UCLM/Spain

CW Speakers:

Haoran Wang

IPHT Bioimaging,Leibnitz Institute, Germany

Maria Vasconcelos

Fraunhofer Institute PT

Abstract:

Reproducible experiments in microscopy demand instruments with a thorough understanding and the capability for independent replication. Open-source Hardware, which involves openly sharing complete designs under accessible licenses, holds the potential to enhance instrument development by fostering reproducibility, accessibility, and mitigating redundant efforts.

Keywords:

open microscopy, reproducibility, light microscopy, quality control

Community workshop

Life cell imaging (CW2)

CW Organizer:

Maria Viñas

Instituto de Óptica/Spain

CW Speakers:

Jana Nieder

INL/Portugal

Mario del Rosario

IGC/Portugal

Abstract:

Bio-Imaging and Biophotonics is a highly interdisciplinary field that exploits the light interaction with biological organisms, tissues, cells and molecules. Live-cell imaging is an important analytical tool in laboratories studying biomedical research disciplines, such as cell biology, neurobiology, pharmacology, and developmental biology. Live-cell microscopy usually involves a compromise between obtaining image quality and maintaining healthy cells. Therefore, to avoid a high illumination intensity and long exposure time, spatial and temporal resolutions are often limited in an experiment. Live-cell imaging is currently mainly performed by fluorescence microscopy, although there is no all-purpose live-cell imaging system suitable for all possible experiments. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope

Keywords:

Cell-friendly; Super-resolution methods; Phototoxicity;

Community workshop

Monitoring and Analyzing Microscope Performance through PSF Measurements (CW4)

CW Organizer:

Nadia Halidi

CRG/SPAIN

CW Speakers:

Nadia Halidi

CRG/SPAIN

Javier Diaz Guerra

CRG/SPAIN

Gabriel Martins

IGC/PORTUGAL

Abstract:

To ensure quantifiable and reproducible microscopy data, it is essential to monitor the performance of the microscope over time. Monitoring performance stability begins with measuring the Point Spread Function (PSF) as an indicator of the optical lateral and axial resolution. The PSF’s size, shape, and symmetry, when compared to the theoretical ideal resolution, provide a comprehensive assessment of the optical setup, including the objective. This, in turn, impacts image quality and the accuracy of subsequent experimental analyses, particularly in advanced microscopy techniques. In this workshop, we discuss and demonstrate how to prepare, acquire tetraspeck beads images and how to analyze the data obtained using an open-source plugin (MetroloJ_QC). We will look at essential metrics such as: Nyquist-Shannon sampling, image quality, shape, size and symmetry of the PSF and the parameters that may influence the shape of PSF.

Keywords:

PSF, monitoring, quality, performance

Community workshop

Cryo-correlative workflows (CW3)

CW Organizers:

Jose Maria Valpuesta

CNB/Spain

Javier Conesa

CNB/Spain

CW Speakers:

Javier Conesa

CNB Spain

Abstract:

Cryo-correlative light and electron microscopy is an integrative and powerful tool for studying biological processes in cells in a fully hydrated state. The combination of spatial and functional information provided by visible light microscopy, along with the high-resolution details obtained through electron microscopy—both performed on the same cell—yields insights that are not accessible through either technique alone. In this workshop, we would like to engage with the scientific community and share our experience with the correlative workflow used to analyze cryopreserved cells. This will include discussions on sample preparation, data acquisition, and data processing.

Keywords:

Cryogenic conditions, correlative light-electron microscopy, TEM, SEM

Community workshop

Optimising STORM technology: Hands-on workflow DEMO (CW5)

CW Organizer:

Ana Mª Oña

CNB/Spain

José Requejo

CNB/Spain

CW Speakers:

Gianluca D’Agostino

CNB/Spain

Jaime Fernandez de Córdoba

CNB/Spain

Abstract:

Stochastic optical reconstruction microscopy (STORM) is a high resolution microscopy technique, developed by X. Zhuang and colleagues in 2006, which allowed to reveal unresolved details of many cellular structures at nanoscale resolution. It is based on the consecutive emission of single photons from the activated state of a photosensitive molecule to allow its precise localization before it enters a dark state or is deactivated by photobleaching. Each fluorophore is activated separately, and by adjusting the point spread function (PSF), the centre of mass can be calculated to determine the location of a molecule down to a resolution of 20 nm. The parallel registration of many individual emitting fluorophores, each with its different set of coordinates, allows the reconstruction of an image with a high degree of resolution [1, 2, 3, 4]. Sample preparation and acquisition conditions require fine-tuning to allow a correct image reconstruction. At the CNB-CSIC Advanced Optical Microscopy Facility, we have optimized a protocol for sample preparation and acquisition conditions that has allowed us to visualize different subcellular structures including the actin cytoskeleton, mitochondria, tubulin, and lysosomes with a resolution in the range of than 50 nm. In addition, we were able to set up an acquisition protocol to perform multicolor STORM for the observation of mitochondria in combination with microtubules, or actin. In the current practical workshop, we want to share with the scientific microscopy community our optimized STORM workflow including crucial steps for sample preparations, live image series acquisition, as well as data processing and image reconstructions using the open source plugin tool ThunderSTORM [5] available for ImageJ. [1] Rust, J.M. et al., Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature methods (2006). [2] Xu, K. et al., Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons. Science (2012). [3] Wang, W. et al., Chromosome Organization by a Nucleoid-Associated Protein in Live Bacteria. Science (2011). [4] Bates, M. et al., Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science (2007). [5] Ovesný, M. et al., ThunderSTORM: a comprehensive ImageJ plugin for PALM and STORM data analysis and super-resolution imaging. Bioinformatics (2014).

Keywords:

STORM, Resolution, Subcellular

Community workshop

Spatial Biology: a game changer(CW6)

CW Organizer:

Diego Megias

ISCIII/Spain

Luisa Cortes

CNC/Portugal

Isabel Peset

CNIO/Spain

CW Speakers:

Diego Megias

ISCIII/Spain

Isabel Peset

CNIO/Spain

Patricia Rodrigues

GIMM/Portugal

Abstract:

Spatial biology is transforming our ability to study complex biological systems by preserving the spatial relationships between cells and tissues. This workshop will focus on advanced microscopy multiplex techniques, including multi-colour immunohistochemistry and cyclic immunofluorescence staining, providing an overview of the different multiplex staining workflows, and will include experimental design, tissue preparation, imaging strategies and advanced image analysis open-source tools. Participants will learn image analysis methods for cell phenotyping, allowing the identification and characterization of cellular subsets within their native tissue environments with applications in cancer, immunology, and neuroscience and will demonstrate how these multiplex and spatial biology techniques are used to decode cellular interactions, tissue microenvironments, and disease mechanisms. Attendees will exchange their understanding of this transformative field and learn how multiplex spatial biology techniques and advanced image analysis can be applied in their research.

Keywords:

Fluorescence multiplex, Spatial analysis, Image analysis, Cell phenotyping

Community workshop

BiaPy: deep learning based Bioimage Analysis for all audiences (CW7)

CW Organizer:

Ignacio Arganda

UPV-EHU/Spain

Edwin Hernandez

UPV-EHU/Spain

CW Speakers:

Lenka Backová

UPV-EHU/Spain

Igancio Arganda

UPV-EHU/Spain

Abstract:

Join us for an engaging workshop on BiaPy, an innovative open-source library designed for versatile bioimage analysis. This session will provide hands-on experience with BiaPy’s user-friendly workflows, zero-code notebooks, and Docker integration, empowering participants to tackle complex bioimage analysis tasks with ease. Whether you are a novice or an experienced developer, discover how BiaPy can streamline your image analysis processes and enhance your research capabilities in life sciences.

Keywords:

Bioimage analysis, deep learning, image segmentation, object detection.