Imagerie-Gif core facility, from our Ile-de-France Sud node, is pleased to announce the acquisition of a Scanning Ion Beam Electron Microscope (FIB-SEM) and a Lattice Structured Illumination Microscope (SIM) Elyra 7. For the occasion, the core facility is organizing “3D Res/volution“, a scientific event on high-resolution 3D imaging on December 15, 2022 from 2:00 pm to 5:00 pm at B21 amphitheatre. This event will be a great opportunity to introduce to you the possibilities of these 2 new systems available at Imagerie-Gif.

Free but mandatory registration: https://evento.renater.fr/survey/3d-res-volution-day-8pdzetbi

Initiated a few years ago, the Inria-IPL-NAVISCOPE (“Image guided NAvigation and Visualization data sets in live cell imaging and microscopy”) project aims at overcoming challenges of bioimaging observation. Virtual and augmented reality could become the new way to visualize and analyze microscope image renders.

Despite incredible progresses in microscopy, imaging biomolecular dynamics in cells remains a challenge. A lack of sensitivity, limited recording speed, photobleaching and phototoxicity associated have restrained, for a long time, our capacity to study biomolecules in their natural environments. As microscopy image is commonly observed on 2D screens, it can narrow human capacities to grasp volumetric, complex, and discrete biological dynamics. Following new modes of visualization including virtual reality (VR)/augmented reality (AR) approaches, the NAVISCOPE project allows more accurate analysis and exploration of large time series of volumetric images, such as those produced by the latest 3D + time fluorescence microscopy.

Why should cell biologists be interested in this project?

The project to which 4 FBI-teams from the BI-IPDM node participate, aims at engineering a technology made with and for biologists. For VR/AR approaches to be adopted by the broader bioimaging community, it is, indeed, important that they are evaluated by the biologists, on their own datasets. 

The potentials of VR/AR technologies for scientists are numerous: navigating into multidimensional, large data sets with another view angle or perception, interacting with these data especially by selecting subregions, quantifying features of interests, etc. New VR/AR approaches also provide specific quantification tools to show distances, angles, counting, local density, and histogram profiler or include a selection of regions of interest for further analysis such as the 3D Timelines. Moreover, because communication with analysis software coded in Java or Python is now integrated, more post-treatment analysis is possible on selected features, providing a multifaceted and accessible tool for biologists.

A promising future ahead

In practice, immersion of the user within 3D + time microscopy data still represents an acculturation challenge for the concerned community. Thus, to promote a broader adoption of these approaches by biologists, further dialogue is needed between the bioimaging community and the VR&AR developers. Nonetheless, future innovation can already be foreseen as there are multiple way to upgrade this technology. For example, using eye-tracking (Günther et al., 2020) or haptic interfaces (Petit et al., 2020) can improve human perception by providing local sensations, which would improve the selection of responses in a 3D + time space. Besides, a better integration of multiple channels with high pixel resolution or the addition of vector representations could add information about the orientation, movement of molecules or organization of structures such as cytoskeleton elements or membrane lipids. The prospects initiated by the NAVISCOPE projects are, as mentioned above, endless and could be a technology that reshapes the way we see biology at the hearth.

Full article on:

Challenges of intracellular visualization using virtual and augmented reality

Valades-Cruz Cesar Augusto, Leconte Ludovic, Fouche Gwendal, Blanc Thomas, Van Hille Nathan, Fournier Kevin, Laurent Tao, Gallean Benjamin, Deslandes Francois, Hajj Bassam, Faure Emmanuel, Argelaguet Ferran, Trubuil Alain, Isenberg Tobias, Masson Jean-Baptiste, Salamero Jean, Kervrann Charleseub

Front. Bioinform. 2:997082.
https://doi.org/10.3389/fbinf.2022.997082

Developed by the Serpico Inria-CNRS-Institut Curie Joint Team, member of the IPDM-BioImage Informatics node of France-BioImaging (FBI), this open-source framework could be a huge step forward in bioimaging management and analysis.

Bioimaging has a broad range of applications, addressing a variety of biological models at diverse scales of life. Thus, descriptions of novel computational approaches are often focused on target case studies. To tackle any scenario in biological imaging is a major challenge, that needs the conception and the development of a unified solution.

With this in mind, the BioImageIT project aims at providing a middleware that integrates data management with analysis using existing softwares (Omero, BioFormats, Fiji, napari, Scipy, pytorch…). The mission of BioImageIT was to design a graphical user interface (GUI) that allows any scientist without coding skills to annotate and analyze datasets using various software. By being user-centered, open-source and cross-platform (Windows, MacOS, Linux), BioImageIT created a management tool that is definitely accessible and well documented.

Started in late 2019, the project, funded by France-BioImaging, is now being deployed in 10 FBI imaging facilities. As it is a first step, the BioImageIT project have the ambition to expand the dissemination of the middleware throughout France and even further, Europe.

BioImageIT overview. a, Schematic view of BioImageIT architecture. The BioImageIT core is composed of data management and data processing functionalities. Users can access plugins by a script editor, Jupyter or the BioImageIT graphical interface (GUI). Data management functionalities exploit local files, remote files or databases such as OMERO. Data processing can perform computations in remote jobs, containers, or local runners. Image analysis is provided by plugins written in different languages. Developers can implement their own plugins in BioImageIT and design their own Graphical Interface. (b-i) LLSM processing workflow gathered in BioImageIT. Hela cell line expressing CD-M6PR-eGFP were stained with Tubulin TrackerTM Deep Red for Microtubules. b, Due to the geometry of LLS scanning, raw 3D images are skewed. c, g, First, realignment (deskew) of raw stacks is performed using Pycudadecon. d, h, Richardson Lucy deconvolution is performed using Pycudadecon. e, CD-M6PR-eGFP vesicles are tracked using Trackmate(FiJi). f, i, Deconvolved stacks and tracks are rendered using napari.

Prigent, S., Valades-Cruz, C.A., Leconte, L. et al. BioImageIT: Open-source framework for integration of image data management with analysis. Nat Methods (2022).
https://doi.org/10.1038/s41592-022-01642-9

Save the date! The Electron Microscopy facility of Imagerie-Gif (I2BC, France-BioImaging), the Cryo-Electron Microscopy facility (I2BC, FRISBI) and the Cimex facility of Ecole Polytechnique are organizing a 5-day workshop from October 3rd to October 7th, 2022 on Transmission Electron Microscopy to explore the architecture of a virus in all its forms.

The aim of this workshop is to propagate knowledge about transmission electron microscopy applications and to outline the advantages of transversal studies combining structural biology and cell biology. Indeed, structural biology and cell biology approaches both use TEM but are rarely merged in the same studies.

The workshop will focus on the advantages of combining both approaches, which can be easily performed with the same equipment. The workshop will focus on a biological object whose study requires such multiscale approaches: a virus. The virus will be studied in vitro to resolve its high-resolution 3D structure, and will be observed in infected cells to determine the infection and replication mechanisms in situ.

The workshop targets students and young researchers. The training will focus on a given biological object, a virus, which will be studied by two complementary approaches:

  • Single particle analysis by cryo-electron microscopy, allowing high-resolution 3D reconstruction of particles purified in vitro. This part will be performed on a 200kV TEM on the Cimex facility.
  • Cellular tomography of infected cells with observation of the virus replication sites in situ and analysis of its interaction with cellular membranes. This workshop will cover the workflow from sample preparation and resin sections realisation, to acquisition and analysis of tomograms with a 120kV TEM.

Attendees will have a theoretical and practical overview of these two complementary techniques. The practical training will be particularly emphasised, to ensure that attendees will be able to apply the knowledge acquired in the workshop for their further research projects.


Susbscription here: https://www.azur-colloque.fr/DR04/inscription/preinscription/245

Preliminary programme

FBI-Programme-3DEM-courseDownload

Understanding how development is coordinated in multiple tissues and gives rise to fully functional organs or whole organisms necessitates microscopy tools. Over the last decade numerous advances have been made in live-imaging, enabling high resolution imaging of whole organisms at cellular resolution. Yet, these advances mainly rely on mounting the specimen in agarose or aqueous solutions, precluding imaging of organisms whose oxygen uptake depends on ventilation.

Engineers from the Institut Curie and Institut Jacques Monod implemented a multi-view multi-scale microscopy strategy based on confocal spinning disk microscopy, called Multi-View confocal microScopy (MuViScopy).

MuViScopy enables live-imaging of multiple organs with cellular resolution using sample rotation and confocal imaging without the need of sample embedding. They illustrated the capacity of MuViScopy by live-imaging Drosophila melanogaster pupal development throughout metamorphosis, highlighting how internal organs are formed and multiple organ development is coordinated. They foresee that MuViScopy will open the path to better understand developmental processes at the whole organism scale in living systems that require gas exchange by ventilation.

3D fusion reconstruction of eight different angles at 10x magnification. Animation of a 3D reconstruction after fusion of images acquired from eight angles by the MuViScope of an Ecad:3xGFP Drosophila pupa at 28 hAPF with a 10x objective. The animation starts with a 180° rotation along the A-P axis and then sequentially shows the eight different angles from a top view: 0° (red), 45° (orange), 90° (yellow), 135° (green), 180° (light blue), 225° (dark blue), 270° (purple) and 315° (pink). Fusion was performed using Huygens Fuser (SVI) and 3D visualization with Imaris software. The acquisition parameters are detailed in Table S1. Scale bar: 200 μm.

Olivier Leroy, Eric van Leen, Philippe Girard, Aurélien Villedieu, Christian Hubert, Floris Bosveld, Yohanns Bellaïche, Olivier Renaud; Multi-view confocal microscopy enables multiple organ and whole organism live-imaging. Development 15 February 2022; 149 (4): dev199760. doi: https://doi-org.insb.bib.cnrs.fr/10.1242/dev.199760

The MuViScope was co-funded by France-BioImaging.

Cryogenic electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. However, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. 

To overcome these obstacles, an international team of scientists from France, Spain and Germany, under the leadership of Charles Kervrann, from France BioImaging BioImage Informatics Node, developed a deep learning-based framework to quickly identify multiple classes of macromolecules in cryo-ET volumes. This DeepFinder pro­gram, now published in Nature Methods, builds upon convolutional neural networks that have already proven highly valuable in the microscopy field.

Overview of DeepFinder (from Moebel, E., et al., Nat Methods 18, 1386–1394 (2021).

a) The DeepFinder workflow consists of a training stage (stage I) and an analysis (or inference) stage (stage II). These two stages correspond to five steps (represented by blue boxes) to locate macromolecular complexes within crowded cells.

b) Ribosome localization with DeepFinder in a cryo-electron tomogram of a C. reinhardtii cell.
Tomographic slice with superimposed segmented cell membrane (gray) and ribosomes classified with respect to their binding states: membrane-bound (blue) and cytosolic (yellow).

c) Tomographic slices showing coordinates
of detected ribosomes (colors correspond to b). The positions and classes were determined by analyzing the segmentation map shown in b. This
analysis used 48 tomograms for training, one for validation and eight for testing. Scale bar, 60 nm.

Once trained, the inference stage of DeepFinder is faster than template matching and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (roughly 3.2 MDa), ribulose 1,5-bisphosphate carboxylase–oxygenase (roughly 560 kDa soluble complex) and photosystem II (roughly 550 kDa membrane complex) with an accuracy comparable to expert-supervised ground truth annotations. DeepFinder is therefore a promising algorithm for the semiautomated analysis of a wide range of molecular targets in cellular tomograms. It also serves as a prime example illustrating the importance of developing efficient, customized AI tools to accelerate knowledge generation in the biomedical life sciences.

DeepFinder has been implemented as a free, open-source program with an accessible graphical user interface.

The team is currently working on adapting it to fluorescence microscopy.

Moebel, E., Martinez-Sanchez, A., Lamm, L. et al. Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms. Nat Methods 18, 1386–1394 (2021). https://doi.org/10.1038/s41592-021-01275-4

Contact
Quantifying translation in space and time during development

During development, precise control of gene expression allows the reproducible establishment of patterns, leading to the formation of organs at the right time and place.

The establishment of developmental patterns has been primarily studied at the transcriptional level. In comparison, the fate of these transcripts received little attention.

Dufourt*, Bellec* et al deployed the SunTag labeling method to image the dynamics of translation of individual mRNA molecules in living Drosophila embryos. This led to the discovery of translation factories and unmasked important heterogeneities in the efficiency of translation between identical mRNAs, demonstrating a novel layer of fine-tuning of gene expression.

Imaging translation dynamics in live embryos reveals spatial heterogeneities.

Dufourt J, Bellec M, Trullo A, Dejean M, De Rossi S, Favard C, Lagha M.

Science. 29 avril 2021 doi: 10.1126/science.abc3483.

Contact:

Mounia Lagha (CNRS)

mounia.lagha@igmm.cnrs.fr

+33 434359653

twitter : drosoigmm

Institut de Génétique Moléculaire de Montpellier (Univ.Montpellier/CNRS) 1919 route de Mende, 34090 Montpellier

For several weeks now, the H2P2 histopathology platform located in Rennes (France BioImaging Bretagne-Loire Node) has become the European reference for the integral solution of Cell DIVE (https://www.leica-microsystems.com/products/light-microscopes/p/cell-dive/).

Cell Dive device on H2P2 platform

This technology brings great expectations for the research teams and the private companies with which we work. Leica’s Cell DIVE technology provides an in-depth solution for characterizing the tissue microenvironment using multiplexed imaging technology. Up to 60 biomarkers can be revealed in one tissue sample. An extensive list of antibodies is already validated and users can customize their own panel! The multiplexed Cell DIVE technology is based on successive immunolabeling of 4 antibodies conjugated with 4 fluorochromes (Cy2, Cy3, Cy5 and Cy7). The slides are digitized (x20 objective) as things progress and a final compiled image is obtained and can be analysed with the Halo Image Analysis Platform. This software allows users to do segmentation to highlight clusters, to define specific cell phenotypes, to analyse neighbourhood, heatmap…

For example, in cancer treatment research, researchers need a better understanding of the cellular architecture of normal and diseased tissues to develop better treatments and more accurately predict disease progression. 

This technology has been developed by scientists for scientists over the last decade and several publications are available to date (https://www.leica-microsystems.com/products/light-microscopes/p/cell-dive/publications/).

Images from human tonsil with 9 biomarkers.

For more information about the Cell Dive technology or to discuss your project, you can contact Nicolas Mouchet nicolas.mouchet@univ-rennes1.fr

Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M

Andrés M. Cardozo Gizzi, Sergio M. Espinola, Julian Gurgo, Christophe Houbron, Jean-Bernard Fiche, Diego I. Cattoni, Marcelo Nollmann

Simultaneous observation of 3D chromatin organization and transcription at the single cell level and with high spatial resolution may hold the key to unveil the mechanisms regulating embryonic development, cell differentiation and even disease. We have recently developed Hi-M, a technology that allows for the sequential labelling, 3D imaging and localization of multiple genomic DNA loci together with RNA expression in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe re-hybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos into microfluidics chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 days including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 days to complete all rounds of labelling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells, or organization of chromatin within complex tissues.

DOI https://doi.org/10.1038/s41596-019-0269-9

Contact: Marcelo Nolmann marcnol@gmail.com

ATP-driven separation of liquid phase condensates in bacteria

B. Guilhas, J.C. Walter, J. Rech, G. David, N.-O. Walliser, J. Palmeri, C., Mathieu-Demaziere, A. Parmeggiani, J.Y. Bouet, A. Le Gall1, M. Nollmann

Liquid-liquid phase separated (LLPS) states are key to compartmentalise components in the absence of membranes, however it is unclear whether LLPS condensates are actively and specifically organized in the sub-cellular space and by which mechanisms. Here, we address this question by focusing on the ParABS DNA segregation system, composed of a centromeric-like sequence (parS), a DNA-binding protein (ParB) and a motor (ParA). We show that parS-ParB associate to form nanometer-sized, round condensates. ParB molecules diffuse rapidly within the nucleoid volume, but display confined motions when trapped inside ParB condensates. Single ParB molecules are able to rapidly diffuse between different condensates, and nucleation is strongly favoured by parS. Notably, the ParA motor is required to prevent the fusion of ParB condensates. These results describe a novel active mechanism that splits, segregates and localises non-canonical LLPS condensates in the sub-cellular space.

Guilhas et al. revealed that the bacterial DNA segregation apparatus behaves as a non-canonical phase separation system. This apparatus employs an ATP-powered motor that splits nanometer-sized condensates and localizes them robustly within the nucleoid to ensure faithful transmission of genetic material.

DOI: https://doi.org/10.1016/j.molcel.2020.06.034

Contact: Marcelo Nolmann marcnol@gmail.com

The ability to communicate effectively with each other is one of the strongest predictors for our chances to get ahead in life. In their latest publication in Science Advances, scientists and engineers from IGF-Montpellier (CNRS, INSERM, Univ. Montpellier), IPAM platform (BioCampus Montpellier, France-Bioimaging Montpellier Node) and ARO-Israel demonstrated that this also holds true for GnRH neurons.

In humans and all vertebrates, species survival depends on a critical step during embryonic development: the migration of a small subset of GnRH neurons (about 2,000 in humans and less than 100 in fish) from the nose to the brain where they join the hypothalamus to control reproduction. Their latest results unveiled that GnRH neurons make a pause at the nose-brain frontier where they function as an inter-hemispheric network that is isolated from the rest of the brain. Only neurons that integrate into the network and are able to communicate with their neighbors will finally cross the barrier and make their way into the brain, towards their hypothalamic destination.

In other words, these GnRH neurons, that are critical for species persistence, face the same challenges like other immigrants: they must learn to communicate effectively if they are to integrate into their new world.

In this study, in vivo 2-photon microscopy was a key tool for:

  • Long term imaging with minimal bleaching and phototoxicity
  • Upright configuration enabling dorsal imaging of the fish in its natural position
  • Long-distance water-immersion objectives allowing imaging of deep tissue structures without sacrificing image quality
  • Fast calcium imaging
  • Imaging of red GECI using the higher wavelengths
  • Precise cell ablation
  • Photoactivation of ChR2 while monitoring Ca in the red channel
A graphical model illustrating the migration of a single GnRH neuron (marked by black border) from the nasal placode into the zebrafish brain.

M. Golan, J. Boulanger-Weill, A. Pinot, P. Fontanaud, A. Faucherre, D. S. Gajbhiye, L. Hollander-Cohen, T. Fiordelisio-Coll, A. O. Martin, P. Mollard, Synaptic communication mediates the assembly of a self-organizing circuit that controls reproduction. Sci. Adv. 7, eabc8475 (2021). doi: 10.1126/sciadv.abc8475

Contact: Patrice Mollard, IGF, Montpellier patrice.mollard@igf.cnrs.fr

Congratulations to Emmanuel Beaurepaire (CNRS Research Director from the Laboratory for Optics and Biosciences CNRS-INSERM-Polytechnique), PI of the ERC Synergy Grant project “HOPE”, and to Laurent Groc (CNRS Research Director ; Interdisciplinary Institute for Neuroscience), coordinator of the ERC Synergy Grant project “ENSEMBLE“, Laurent Cognet (CNRS Research Director ; Laboratoire photonique numérique et nanosciences) and U. Valentin Nägerl (Professor at University of Bordeaux ; CNRS Research Director ; Interdisciplinary Institute for Neuroscience), both PIs of the ERC Synergy Grant project “ENSEMBLE“.

These grants, each worth around 10 million euro over six years, are designed to enable groups of 2 to 4 scientists to tackle some of the world’s most challenging research problems, spanning several scientific disciplines.

The ERC Synergy Grant scheme is part of the EU’s research and innovation programme, Horizon 2020.

ERC Synergy Grant project “HOPE”

“Reverse engineering the assembly of the hippocampal scaffold with novel optical and transgenic strategies” 

  • Emmanuel Beaurepaire, Directeur de recherche CNRS au Laboratoire d’optique et biosciences – LOB (CNRS/École polytechnique/INSERM),
  • Rosa Cossart, Directrice de recherche CNRS (Unité INSERM, Aix-Marseille Univ.)
  • Jean Livet, chercheur INSERM à l’Institut de la vision (CNRS, INSERM, Sorbonne Univ.)

At the heart of our brain, a structure plays a key role in memory, and more particularly in the acquisition and maintenance of our memories: the hippocampus. Classically considered as a “cognitive GPS” for space and time, it is also the seat of our episodic memory.

Over the last decade, the neural circuits of the hippocampus have been better described, in particular by the team of Rosa Cossart, director of the Institut de neurobiologie de la méditerranée (Inmed), but the nature, origin and remodeling of these circuits during development and pathologies remain to be understood.

On the other hand, genetic engineering techniques for staining neurons, developed by Jean Livet, Inserm research director at the Institut de la vision, coupled with multi-photon microscopy developed by the team of Emmanuel Beaurepaire, CNRS research director at the Laboratoire d’optique et biosciences – LOB (illustration below / read the 2019 press release in French), have demonstrated their ability to accurately map the complex architecture of neuronal circuits and their evolution during development.

By combining these exceptional multidisciplinary advances, HOPE aims to answer three interdependent questions:

  • Is the architecture of the adult seahorse carried by specific circuits?
  • Are the circuits of the hippocampus pre-wired or shaped by experience?
  • How does this structure reorganize itself in pathological conditions?

HOPE aims to shed new light on the function of the hippocampus and the role of its neuronal circuits through the design of a new, non-invasive and universal method to monitor the growth and construction of brain circuits located deep in the brain, from their neurogenesis to adulthood, under normal and pathological conditions.

Developed by the LOB team, ChroMS is a new microscopy technique combining color, 3D and high resolution, introducing a real revolution in vertebrate brain imaging. © Lamiae ABDELADIM / LOB / Institut de la Vision / CNRS Photothèque

Taken from CNRS Press release: https://www.iledefrance-gif.cnrs.fr/fr/cnrsinfo/erc-synergy-grant-2020-3-projets-impliquant-le-cnrs-sur-le-territoire-paris-saclay

ERC Synergy Grant project “ENSEMBLE”

“Structure and functions of the brain extracellular space“

  • Laurent Groc (Research Director CNRS ; Interdisciplinary Institute for Neuroscience), 
  • Erwan Bézard (Research Director INSERM; Institute of Neurodegenerative Disorders), 
  • Laurent Cognet (Research Director CNRS ; Laboratoire photonique numérique et nanosciences)
  • U. Valentin Nägerl (Professor at University of Bordeaux ; Research Director CNRS ; Interdisciplinary Institute for Neuroscience)

The ENSEMBLE project aims at underpinning the molecular mechanisms of physiological and pathological brain function. This ambitious and innovative endeavor is based on our ability to develop new approaches in high-resolution microscopy at the service of a new conceptual framework in brain cell communication.

Brain, high resolution iimage. Credit: U.V. Nägerl
Credit: U.V. Nägerl

This project has roots in the international leadership of the Bordeaux community in the fields of microscopy, nanophotonics, fundamental and translational neuroscience. The opportunity that is offered to these 4 investigators to break a frontier knowledge was permitted by the continuous support of local institutional actors. The installation of Prof. Valentin Nägerl’s laboratory in 2009 with a “Chaire Accueil” from the Regional Council of Aquitaine, the support of LabEx BRAIN, the Laphia Cluster and the IdEx of the University of Bordeaux provided the ground to build elementary blocks necessary for the challenging adventure of the ERC Synergy project (10 million euros, 6 years).

Taken from Bordeaux Neurocampus Press release: https://www.bordeaux-neurocampus.fr/en/erc-synergy-award-2020-for-groc-bezard-nagerl-and-cognet/