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:

  • Technology transfer: spot variation Fluorescence Correlation Spectroscopy (svFCS)

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:

  1. 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).
  2. Germani, E., Lelouard, H., Fallet, M. SAPHIR: a Shiny application to analyze tissue section images, F1000Research, Faculty of 1000, 9, 1276-1285 (2020).
  3. 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)

Congratulations to Patrick LEMAIRE, CNRS Research Director from the
Centre de recherche en biologie cellulaire de Montpellier (CRBM) – CNRS / Université de Montpellier, member of France BioImaging Montpellier Node, who has been awarded the Mottart prize.

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.

To learn more about the study: https://france-bioimaging.org/announcement/news-from-nodes/montpellier-node-participated-on-a-study-of-the-dynamics-of-cell-movement-during-embryonic-development/

COMULIS is an EU-funded COST Action that aims at fueling urgently needed collaborations in the field of correlated multimodal imaging (CMI), promoting and disseminating its benefits through showcase pipelines, and paving the way for its technological advancement and implementation as a versatile tool in biological and preclinical research. CMI combines two or more modalities to gather holistic information about the same specimen. It creates a composite view of the sample with multidimensional information about its macro, meso- and microscopic structure, dynamics, function and chemical composition. Since no single technique can reveal all these details, CMI is the only way to understand biomedical processes mechanistically.

In order to encourage correlated multi-modal imaging projects, COMULIS Short Term Scientific Missions (STSMs) provides travel grants to individuals wishing to explore new imaging techniques. The grants are presented in the form of a lump sum of up to 3,500 Euros (depending on the duration of the mission), to cover travel and subsistence. COMULIS COST accepts applications on a continuous basis from Early Career Investigators and Experienced Imaging Scientists who would like to travel internationally to collaborate with a Host facility on a Correlated Multi-modal imaging project. There is a rapid review process and around 10 grants are awarded every year.

In addition, COMULIS STSM can provide funding for core facility staff to learn a new imaging  technique or work with new software tools to bring the expertise back to their own facility.

Applications can be submitted any time & will be reviewed at the end of each month.

More info and application procedure: https://www.comulis.eu/stsm-open-call

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!

Agave software:
https://www.allencell.org/software-and-code.html
https://www.allencell.org/pathtrace-rendering.html
Article (in French):
https://www.science-et-vie.com/corps-et-sante/regenerer-le-cerveau-des-cellules-souches-retablissent-les-liaisons-neuronales-p-58266#dossier-58457

During embryonic development, cells take on increasingly precise roles in the body as they divide. Be they skin cells, muscle cells or neurons, the different cell types that make up the embryo emerge gradually from a very fine orchestration of their positions and identities, coordinated by the signals they exchange with each other. Like us, the cells need to “talk” to each other to make decisions.

Screaming or whispering: the embryonic cell dilemma

In vertebrate embryos, cells have a very dynamic behaviour. They move around, exchange their neighbours or migrate over long distances. The signals they exchange therefore need to have a long range, which could be characterized as “shouting”. The study of the embryonic development of a sea squirt, a small marine animal with optically transparent embryos, has enabled scientists from several teams at CNRS and INRIA in France, in collaboration with a team from the European Molecular Biology Laboratory (EMBL, Germany), to capture and describe in detail a more discreet mode of cell communication.

The scientists recorded the development of live embryos every two minutes with a new-generation « light-sheet » microscope. They then created software to automatically detect each cell and analyze its position, shape and neighbours up to an advanced stage of development. This work revealed an unusually reproducible mode of development, in which the same cell can be found in the same position across all embryos and where cells move very little in relation to each other. The authors of the study then annotated the films thus made with information on the cell type and the molecular signals emitted by each cell. Using mathematical modelling to integrate the quantitative description of the embryonic geometry with these annotations, their work suggest that cells communicate with very short-range signals. Moreover, the interpretation of these signals is modulated by the area of the contacts between cells. Unlike vertebrates, the cells of ascidian embryos thus have a static and fixed behaviour and the range of their “whispered” signals is very small.

Top: embryonic development of an ascidian from egg to tadpole. The part framed in white is the part of embryogenesis that we have imaged and then segmented (below, segmented cells coloured according to their cell fate). The lower part of the figure illustrates that the light green cells “whisper” instructions to their immediate neighbours by short-range signals.

This study indicates that the dynamics of cell movement varies greatly between animals and that these different modalities could be strongly related to the range of signals that the cells exchange with each other. By extending the repertoire of cellular communication mechanisms, this work opens new perspectives on the understanding of self-organization strategies of living forms.

Article: L. Guignard*, U.-M. Fiuza*, B. Leggio, J. Laussu, E. Faure, G. Michelin, K. Biasuz, L. Hufnagel, G. Malandain, C. Godin#, P. Lemaire# (2020) Contact-area dependent cell communications and the morphological invariance of ascidian embryogenesis (Science, July 10 2020 issue, https://science.sciencemag.org/content/369/6500/eaar5663)

Guignard-et-al.-2020-Contact-area–dependent-cell-communication-and-the-morphological invariance of ascidian embryogenesisDownload

2 recent publications using the laser irradiation and photoablation systems available on the MRic facility from the Bretagne-Loire Node are presented here:

  • Esmangart de Bournonville and Le Borgne (IGDR) characterized the assembly and interactions of tricellular junction components in Drosophila epithelial cytokinesis using laser ablation on a SP5 confocal. Their article entitled “Interplay between Anakonda, Gliotactin, and M6 for Tricellular Junction Assembly and Anchoring of Septate Junctions in Drosophila Epithelium” was published last august in Current Biology (https://doi.org/10.1016/j.cub.2020.07.090).
  • Rebecca Smith, post-doc in Sébastien Huet’s team (IGDR) in collaboration with Szilvia Juhász from Gyula Timinszky’s team (Szeged, Hungary) used laser irradiation to study chromatin remodeling following DNA damage. Their paper entitled “The chromatin remodeler ALC1 underlies resistance to PARP inhibitor treatment” has just been accepted in Science Advances.

During this year 2020, the MicroPICell facility from the Bretagne Loire Node acquired several imaging systems, some of which offer access to new technologies on the Nantes health research site:

  • a complete Zeiss Lighsheet 7 light sheet microscope associated to an X-Clarity clearing system and an Arivis Vision 4D Offline station,
  • a motorized Nanolive holotomographic microscope,
  • a high-end Nikon confocal microscope (resonant, spectral, FLIM, large field of view),
  • an Akoya CODEX system of multiplex fluorescent tissue labeling.
Holography offers a unique means to measure cells in their native environment: label-free, non-invasive, manipulation-free, and interference-free.

Moreover, the MicroPICell facility, in collaboration with the training organization of the CNRS, is organizing in March 2021 a training on histology: from sample preparation to markers validation by image analysis. This training (lectures, workshops) will take place over 4 days between 03/22/2021 and 04/24/2021.

Link: https://cnrsformation.cnrs.fr/stage-21290-Histologie–de-la-preparation-dechantillons-a-la-validation-des-marquages-par-analyse-dimage.html?stage=21290&axe=138

Congratulations to Emmanuel Beaurepaire, CNRS Research Director from the Laboratory for Optics and Biosciences CNRS-INSERM-Polytechnique, and member of France BioImaging Ile de France Sud Node, who has been awarded the 2020 Life Sciences prize from the European Microscopy Society for his outstanding achievements in: 

“the fields of developmental biology and neurobiology by development of novel, cutting-edge light microscopy techniques. Notably, he pushed forward methods of deep-tissue imaging, with important application potential for insights into developing small model systems and for brain imaging, using advanced techniques such as multicolor two-photon excitation, third-harmonic generation imaging, adaptive optics, pulse shaping, etc. “

On August 27th, 2020, Emmanuel presented his work during the award ceremony during the EMS General Assembly, which has been held by visioconference this year.    

The Global BioImaging Exchange of Experience workshop series continues with an online event on “Pre-publication image data: management and processing” which will be held on September 8th and 9th, 2020!

Global BioImaging and ABiS will host a two-half days virtual meeting, featuring high-level speakers from around the world and introducing the topic to the global community. A second meeting is planned, where GBI hope to be able to gather the community in person in Okazaki in spring 2021 and continue fruitful discussions and the scientific networking of our community.

As partner of the Global BioImaging initiative, France BioImaging encourage the FBI community to participate in this 2 half-days virtual meeting and share inputs on solutions related to the management of image data before they reach the publication stage!

Registrations are now open: please follow this link and register!

More information on the event can be found here: EoE V webpage.

Congratulations to Pierre-François Lenne, CNRS research director and group leader at the Institute for Developmental Biology of Marseille-Luminy (IBDM), who was elected member of EMBO. His current research focuses on cell dynamics and mechanics in the context of tissue morphogenesis.

Pierre-François is also FBI Marseille Node representative and scientific manager of the FBI Picsl imaging facility at the IBDM.

EMBO announced the list of newest elected members on July 7th, 2020.

EMBO’s tradition of recognising outstanding life scientists as Members dates back to 1963, when an initial group of 150 Members were selected by EMBO’s Council. Since then, EMBO Members have been invited to nominate and elect exceptional researchers to join the community, which now exceeds 1,800 Members and Associate Members. Elections for EMBO Members are held annually. The new EMBO Members join a growing list of renowned researchers elected before them, which includes 88 Nobel laureates.

“The new Members have contributed to the success of research in the life sciences in Europe and around the world,” said EMBO Director Maria Leptin. “As EMBO Members they can help to shape the future through EMBO’s work to support talented researchers, bring ideas together, and promote an international research environment conducive to excellent science.”

EMBO Members actively participate in EMBO initiatives, for example by serving on EMBO Council and committees, by mentoring young scientists, or supporting activities such as the promotion of sound science policy. Members also guide and support the organisation in ensuring the highest quality in the selection of future members, postdoctoral fellows, and courses and workshops.

Source: EMBO press release

More info on EMBO: www.embo.org

CZI’s new Deep Tissue Imaging RFA aims to advance the field of deep tissue imaging and support the development of technologies that will allow researchers to view information at cellular resolution, in complex tissue and through skin and bone, in living organisms. CZI invites scientists to apply for this 2 1/2-year grant opportunity, and grants will be for $1 million in total costs per grantee. 

In the first phase of the RFA, grantees will develop a pilot project. Successful outcomes could include the development of imaging technologies and biological probes needed to visualize and label important cellular processes throughout the body, or new computational techniques and algorithms for deep tissue signal extraction and analysis. In the second phase of the RFA, successful grantees will be eligible to apply for four-year, $10 million technology development grant awards. Final determination of awards and numbers will depend on the quality of the applications received. 

Scientific Scope

The long-term goal of this initiative is to drive technology development aimed at visualizing cellular structure and function throughout the body. During this pilot phase, they especially seek proposals that support the development of tools for visualizing cellular level processes in deep tissue. This funding opportunity is explicitly aimed at technology development. It is not intended to support question-driven basic or translational research, clinical trials, or drug development.

Examples of research themes:

·         Bioacoustic probe, hardware, and/or method development

·         Biomagnetic probe, hardware, and/or method development

·         Biochemical probe or method development

·         Multi-photon hardware or method development

·         Deep imaging tissue techniques with potential human applicability

The Deep Tissue Imaging RFA will accept Letters of Intent starting Thursday, July 9, 2020 at 9 a.m. Pacific time until Thursday, August 6, 2020 at 5 p.m. Pacific time. For more information and application instructions, please visit CZI’s online grants management portal. For administrative and programmatic inquiries, technical assistance, or other questions pertaining to this RFA, please contact sciencegrants@chanzuckerberg.com. Learn more about CZI’s Frontiers of Imaging effort.

CZI’s Imaging Scientists Cycle 2 RFA is also currently open until July 30, 2020 at 5 p.m. Pacific Time. Read more about CZI’s Imaging program.

More information about CZI’s Deep Tissue Imaging RFA: https://chanzuckerberg.com/rfa/deep-tissue-imaging/