Publication: Multi-view confocal microscopy enables multiple organ and whole organism live-imaging
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Category: News from Nodes
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 Methods18, 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 Methods18, 1386–1394 (2021). https://doi.org/10.1038/s41592-021-01275-4
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.
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.
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.
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.
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.
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 theERC 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 theERC 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.
“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.
Credit: U.V. Nägerl
This project has roots in the international leadership of the Bordeaux communityin 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).
We are very pleased to announce that FBI Bretagne-Loire Node application to become a Euro BioImaging facility has been evaluated as highly recommended by the EuBI Scientific Advisory Board (SAB) and ratified by the EuBI Board on November 30th, 2020.
The Bretagne Loire Node became a node of the national infrastructure France BioImaging in November 2019 and applied to become a EuBI facility during the last EuBI Call for Nodes (June 2020).
The Bretagne Loire Node brings together four cellular imaging and histology facilities, two in Rennes (MRic and H2P2) and two in Nantes (MicroPIcell and APEX). These facilities have complementary expertise for live imaging and pathological anatomy. The added value of the Bretagne Loire Node is to be able to offer a continuum between biological imaging and medical imaging, through the development of a new line of services as well as methodological and technological transfer to users of microscopy technologies for preclinical studies.
As part of the Euro-BioImaging research infrastructure, the services provided by these facilities will be open to all scientists, regardless of their discipline or affiliation.
The PICsL-FBI microscopy core facility is located on two sites: Centre d’Immunologie de Marseille Luminy (CIML) and Institut de Biologie du Développement de Marseille (IBDM).The PICsL-FBI facility of the CIML called ImagImm (Imaging Immunity) via its microscopy resources – from the molecule to whole organisms – is dedicated to help its users deciphering cellular mechanisms in the fields of immunology.
Major research implications of the ImagImm facility:
In collaboration with Tomasz Trombik (Faculty of Biotechnology, University of Wroclaw – Wroclaw, Poland), Sophie Brustlein (Institut de Convergences Centuri, AMU,CNRS – Marseille, France) and Nicolas Bertaux (Institut Fresnel, AMU, Centrale Marseille, CNRS – Marseille, France), Sébastien Mailfert and Didier Marguet published the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized1. This publication is following the technology transfer made in 2018: the svFCS developed by Didier Marguet’s lab was duplicated by Sébastien Mailfert and Sophie Brustlein and built from scratch in 7 days on site, in Poland.
Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. The published protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment is provided and how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. This fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.
Figure 1:Schematic view of excitation and emission optical paths of the svFCS setup and pictures of the setup. The svFCS setup contains four modules: (1) the output of a fibered 488 nm laser is collimated, (2) a combination of a half-wave plate and polarizing beamsplitter sets the optical power, (3) the laser beam focused on the sample after traveling through a tube-lens free motorized microscope, and (4) the fluorescence is detected through a confocal-like detection path onto an avalanche photodiode coupled to a single photon counting module, which delivers a signal to a hardware correlator. Simplicity gives the system its sensitivity, robustness, and ease of use.
SAPHIR : a Shiny application to analyze tissue section images
In collaboration with Hugues Lelouard (CIML, Inserm, CNRS, AMU) and Elodie Germani, Mathieu Fallet published a powerful method for both basic and medical research to study cell populations in tissues using immunofluorescence. Image acquisitions performed by confocal microscopy notably allow excellent lateral resolution and more than 10 parameter measurement when using spectral or multiplexed imaging. Analysis of such complex images can be very challenging and easily lead to bias and misinterpretation. They developed the Shiny Analytical Plot of Histological Images Results (SAPHIR), an R shiny application for histo-cytometry using scatterplot representation of data extracted by segmentation. It offers many features, such as filtering of spurious data points, selection of cell subsets on scatterplot, visualization of scatterplot selections back into the image, statistics of selected data and data annotation. This application allows to quickly characterize labeled cells, from their phenotype to their number and location in the tissue, as well as their interaction with other cells.
Figure 2: Flow chart of tissue image analysis from image acquisition and segmentation (left side) to extract data analysis with SAPHIR (right side)
Wound healing in C. elegans
In collaboration with Nathalie Pujol and Jonathan Ewbank (CIML, Inserm, CNRS, AMU), Mathieu Fallet and Sébastien Mailfert participated in the project on the immune response by showing that wounding provokes a reorganization of plasma membrane subdomains3. The skin protects animals from infection and physical damage. In Caenorhabditis elegans, wounding the epidermis triggers an immune reaction and a repair response, but it is not clear how these are coordinated. Previous work implicated the microtubule cytoskeleton in the maintenance of epidermal integrity (Chuang et al., 2016). Taffoni et al. show the reorganization of the plasma membrane subdomains by a simple wounding system. This is followed by recruitment of the microtubule plus end-binding protein EB1/EBP-2 around the wound and actin ring formation, dependent on ARP2/3 branched actin polymerization. They show that microtubule dynamics are required for the recruitment and closure of the actin ring, and for the trafficking of the key signaling protein SLC6/SNF-12 toward the injury site. Without SNF-12 recruitment, there is an abrogation of the immune response. These results suggest that microtubule dynamics coordinate the cytoskeletal changes required for wound repair and the concomitant activation of innate immunity.
Figure 3: Time line of events
References:
Mailfert, S., Wojtowicz, K., Brustlein, S., Blaszczak, E., Bertaux, N., Łukaszewicz, M., Marguet, D., Trombik, T. Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells, JoVE, 165, 1-19 (2020).
Germani, E., Lelouard, H., Fallet, M. SAPHIR: a Shiny application to analyze tissue section images, F1000Research, Faculty of 1000, 9, 1276-1285 (2020).
Taffoni, C., Omi, S., Huber, C., Mailfert, S., Fallet, M., Rupprecht, J-F,. Ewbank, J., Pujol., N. Microtubule plus-end dynamics link wound repair to the innate immune response, eLIFE, 9, e45047 (2020)
With his team members, Patrick Lemaire is studying the embryonic development of a small marine invertebrate, the sea squirt Phallusia mammillata, chosen for the simplicity and transparency of its embryos. His latest work has combined microscopy, image analysis and mathematical modeling approaches to describe, cell by cell, the embryogenesis of this animal and to analyze the role of communication between cells.
The image depicts a spheroid of human stem cells (green) and its actin cytoskeleton (purple), produced by Philippe Cohen during its PhD at Treefrog. This nice picture serves as an illustration for an article covering the use of stem cells for regenerative medicine. Acquisition was made by Philippe Cohen on a scanning confocal microscope and 3D rendering was done by Jérémie Teillon using Agave software. Agave is a free 3D visualization software, using light path-trace light rendering.
The Bordeaux Imaging Center team offers training and support on 3D commercial softwares such as Imaris and Arivis as well as other freeware such as Agave. Don’t hesitate to contact them (bic@u-bordeaux.fr) if you are interested in 3D rendering and visualization of your microscopy data!
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