Researchers and imaging engineers know better than anyone that an image is always more than meets the eye. Let us honor the winning image of our 2017 Image Contest by delving a little deeper into what lies behind these Venice canals.

The image represents a large part of mice mammary gland. The canals spreading out like branches, are composed of two layers of cells: the epithelial epithelial cells – stained by anti-keratin 8 (in pink) – are surrounded by myoepithetial cells (in blue) stained by an anti-Smooth Muscle Actin. These cells, thanks to their contractile properties, participate in the? secretions from the gland. The yellow cells are modified cells, overexpressing tomato protein. The adipocytes of the mammary gland can be seen in the background.

The image was made by Marie Irondelle (PICT-IBiSA Biomaging Cell and Tissue Core Facility, Curie Institute), using a confocal microscope. It is a mosaic reconstruction – using the tile scan technique -from several depths of field with a range of 110 µm. The lighter areas are higher up in the sample, while the darker areas correspond to zones deeper in the tissue. The final projection measures 1.2 by 0.78 mm. The ducts shown in the picture have a diameter of 50 to 60 microns.

Carine Rossé and Emilie Lagoutte in the lab of Philippe Chavrier (membrane & Actin dynamics lab), from the Curie Institute, set out to study the behavior and movement of few cancerous cells inside the mammary gland. However, such a study required the development of alternative methods of observation, from the traditional methods of analysis, in order to observe the localization of the cells in the whole gland. The solution came from a 2016 Nature Methods paper entitled “Shrinkage-mediated imaging of entire organs and organisms using uDISCO”[1]. In this method, the researchers described the advantages of the uDISCO tissue-clearing protocol for the analysis of large samples, compared to other well-known methods. After adapting the protocol, Emilie Lagoutte was able to clear entire mice. She obtained and stained mammary glands that were shrunk by about 70% compared to their original size, cleared. The advantages of this technique are numerous; in particular, there is no more tissue loss due to slicing samples, the fluorescence can be maintained for a few weeks, and the reduced size of the sample allows to visualize the whole organ, leading to a higher chance to detect the zone of interest.

Imaging facilities and researchers have to work hand-in-hand to produce the best results possible. Marie Irondelle stressed the necessity for researchers to be educated about imaging technologies and their limitations; a state-of-the-art microscope will never be able to compensate for a low-quality sample. In the case of The Little Venice, the PICT-IBiSA Biomaging Cell and Tissue Core Facility collaboration with the Chavrier lab is undoubtedly a winning one.

[1] https://www.nature.com/articles/nmeth.3964

This year, the Focus on Microscopy Conference took place in sunny Bordeaux, France, from April 9th to April 12th. France BioImaging participated in two ways: with a booth in the exposition hall, and through a symposium co-organized with Euro BioImaging, held on Wednesday 12th.

The France BioImaging Booth

Many curious attendees stopped by our booth to enquire about our activities. In particular, foreign visitors were curious to know how the infrastructure was organized, as they expressed the intention to set up similar services in their own countries. Industrials were equally intrigued by our organization, and expressed the desire to work with us towards the dissemination of their new available technologies.

Volunteers from the Bordeaux Node gracefully assisted the coordination team in setting up & animating our booth.

Symposium on Training

With more than 40 attendees, the Symposium on Training (Wednesday 12 April) was a great success. The public and speakers shared fruitful ideas about the past, present and future of biological imaging training at the European level.

You can find the program of the Symposium here.

 

We would like to thank the Focus on Microscopy organizers for their work in ensuring the success of the conference.

Our 4th Annual Meeting took place on Friday April 14th 2017. We had the pleasure of welcoming about 80 participants at the Curie Institute. This year, the meeting focused on the question of future challenges in biological imaging.

The four sessions of the program were centered around the following topics:

• Quantification of the molecular dynamics and coordination in cells and small organisms, including at the nanometer scale
• Imaging architectures and processes of life, from molecular complexes to multi cellular systems
• New frontiers for imaging, sensing, and controlling biomolecules
• Bioimage informatics, image processing and microscopy data management

At lunch time, the room was buzzing during the poster session.

We would like to thank our invited speakers, Teng-Leong Chew, Enrico Gratton, Markus Sauer & Jean-Christophe Olivo-Marin, as well as our chairs & all participants who shared their work with their peers through a talk or a poster.

Find all the details of the organization & program here.

The FBI Marseille node is composed of the PICsL light and electron microscopy core facility and three associated research and development (R&D) labs. The FBI-PICsL facility is located on the Marseille Luminy campus and hosted by two institutes: the Centre d’Immunologie de Marseille Luminy (CIML) and the Institut de Biologie du Développement de Marseille (IBDM). The R&D labs comprise two labs of these institutes (Lenne lab at IBDM and Marguet lab at CIML) and the biophotonics lab of the Fresnel institute (Mosaic group led by H. Rigneault).  The FBI-PICsL facility via its microscopy resources is dedicated to help its users deciphering cellular mechanisms in the fields of cell biology, immunology and developmental biology.

In 2017, the FBI-PICsL facility comprises 40 setups. Altogether, these microscopes cover a wide range of temporal and spatial scales from the second to milliseconds and from single molecules to entire organisms. This repertoire of microscopes is composed of cutting-edge light microscopes (FCS, FCCS, STED 3D, PALM/dSTORM, Fast polarization-resolved spinning disk, Light sheet microscopes) among which 8 are homemade and 3 electron microscopes (two TEMs and one SBF-SEM). Ten staff members manage the facility under the scientific supervision of Pierre-François Lenne (IBDM) and Didier Marguet (CIML). The FBI-PICsL facility staff is also involved in teaching microscopy techniques to the facility users and within courses open to external academic and industrial users.

New set-ups on the FBI-PICsL facility

LSM 880 Fast-Airyscan

A new confocal microscope using the technology Airyscan (LSM 880 Fast-Airyscan, Zeiss) has been purchased through a co-funding FBI/Inserm/CNRS/Region PACA. The Airyscan improves spatial resolution (1.7 x) by imaging the Airy disk onto a concentrically arranged hexagonal detector array. Its detection area consists of 32 single detector elements, each of which acts like a very small pinhole. The confocal pinhole itself remains open and doesn’t block light – thus all photons of the whole Airy disk are collected. The signals from all detector elements are then reassigned to their correct position, producing an image with increased signal-to-noise ratio and resolution.

FEI Teneo VS

The FBI-PICsL facility also recently homed an electron microscope dedicated to automated 3D imaging.

Revealing the complex 3D architecture of cells and tissues is crucial for the structure-function correlation in biological systems. However, in most of the electron microscopy experiments, only a small portion of the sample is imaged. For instance, one 70nm-section of an average vertebrate cell only represents 0.4-1.0% of its volume. Beyond the fact that this tiny volume might not be representative to account for the entire cell ultrastructure, the sectioning also avoids a definite visualization of the cellular organization.

Until recently, the imaging of big volumes (tens to hundreds of cubic micrometres) by electron microscopy was tedious because it involved manual serial sectioning. Beyond the need of super-skilled people, manual serial sectioning has a limited z resolution of 70nm and is a time-consuming process at the sectioning, imaging and post-processing steps. Within the last 10 years, the Serial Block-Face Scanning Electron Microscopy (SBF-SEM) has emerged as the method of choice for the imaging of big volumes by electron microscopy.

The concept behind SBF-SEM is to have a microtome within the chamber of a scanning electron microscope (see Figure). This set-up enables the iterative sectioning and imaging of the sample block-face for hundreds of microns in a fully automated way (Figure). When an electron beam hits a sample, it interacts with it and yields several signals that can be detected and assigned back to the scanned positions on the sample surface. Among these signals, the backscattered electrons can be collected to retrieve information on the sample electron density at a given point. Routinely, a working pixel size in SBF-SEM datasets is ±5nm and the slicing can be as thin as 20-30 nm.

In addition to be an excellent microscope in classical SEM mode, the Teneo VS microscope by FEI is currently the most advanced microscope to carry out SBF-SEM. It does not only allow you to cut and image the sample in a fully automated way but it also gives the possibility to avoid charging artefacts during imaging by putting the sample in low vacuum conditions. By limiting the charging, the microscope gives you the opportunity to retrieve more information from the sample. For instance, the sample can be scanned with different acceleration voltages in order to retrieve ultrastructural information at different depths (10nm – 20nm – 30nm – 40nm). When this multi-energy imaging is combined to a 40nm mechanical slicing, it gives rise to previously inconceivable datasets of hundreds of cubic microns of tissue with a voxel size of 5x5x10nm !

Since the delivery of the FEI Teneo VS microscope at the FBI-PICsL facility in December 2016, we tested the microscope on a broad range of biological samples in classical SEM mode (fly egg-laying apparatus, Xenope embryo, mouse face…) and in SBF-SEM mode (cell pellets, Drosophila muscle, mouse brain, marine sponges, Xenope embryo…). In particular, the group of Laurent Kodjabachian (IBDM), working on multiciliated cells, wanted to image the same cell type in both classical SEM mode and in SBF-SEM. We could 1. assess the penetrance of a mutant phenotype in classical SEM mode and 2. localize the basal bodies of these multi-ciliated cells in SBF-SEM (see Figure below).

Photo credits: Virginie Thome and Nicolas Brouilly

Major implications of the FBI-PICsL facility

Spot variation fluorescence correlation spectroscopy (svFCS) (Hai-Tao He and Didier Marguet’s team)

In collaboration with Nicolas Bertaux (Institut Fresnel, AMU, Centrale Marseille), members of the Hai-Tao He and Didier Marguet team published a user guide for characterizing plasma membranes subdomains in living cells by sv-FCS2. This book chapter aims to serve as a guide for setting and applying the svFCS methodology to study the plasma membrane of both adherent and nonadherent cell types.

 

This technique has been used to understand a genetic disease by studying molecular diffusion at the plasma membrane. In collaboration with Christophe Lamaze (Institut Curie) and Céline Galès (Institut des Maladies Métaboliques et Cardiovasculaires de Toulouse), Hai-Tao He and Didier Marguet’s team worked on decoding the dysfunction of molecular mechanisms involving interferon-γ (IFN-γ), key protein for immune defense. This protein is not able to fulfil its natural function as a protective cytokine when its receptor is “blocked” in the wrong place on the cell membrane: a modification to the immune response causing severe infections sometimes even life-threatening for young children.

IFN-γ receptor is composed of two protein chains to which are generally associated six sugars. However, some patients diagnosed with Mendelian susceptibility to mycobacterial diseases syndrome (MSMD) have seven sugars. This simple gain of glycosylation affects greatly the immune response against mycobacteria.

Using their expertise in biophysics and more particularly in svFCS, this CIML team showed the existence of nanodomains and their fundamental role in signaling activity. “It was completely unexpected that only one additional sugar could modify the way the receptor is localized on the surface of the cells, affecting greatly the later signaling events”, explained Hai-Tao He. “IFN-γ receptor is then delocalized towards another membrane nanodomain populated by galectin proteins, that bind to this additional sugar” added Yannick Hamon2 (cf Figure)

As therapeutic target, galectins are an appealing perspective, particularly as turning off their expression canceled in vitro the pathologic effect linked to the glycosylation gain of the IFN-γ receptor. However, therapeutic programs towards patients requires the exploration and understanding of molecular mechanisms by uniting expertise of biologists and physicians but also mathematicians and physicists as it was the case in this discovery4.

News

In March 2017, a workshop on super-resolution is organized in Marseille by Sébastien Mailfert, Jean-Bernard Fiche and Orestis Faklaris on multicolor STORM an dSPT-pointillism data analysis, this workshop is dedicated to advanced researchers in this field. This event is supported by FBI.

The FBI-PICsL facility is also homing the 14th RTmfm convention from the 20th to the 22nd of March 2017. This convention is a great opportunity for imaging facilities staff to share their experiences and ideas on the past, present and future of their work (technical development, big data issues and solutions, link with industry).

Our facility also yearly organizes the following course: Scientific platform, instrumental sharing, how to build up and develop a service. The aim of the course is to acquire, by alternating courses, exercises and practical conditions, methods and constraints to the development of a provision of scientific equipment service. We insist on how to develop a contributory model for managing a scientific platform.

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References

1. Mailfert, S., Hamon, Y., Bertaux, N., He, H-T. & Marguet, D. Methods in Cell Biology, 139, 1-22 (2017)
2. Blouin, C., Hamon, Y. et al. Cell, 166, 920-934 (2016)
3. http://www.cell.com/cell/fulltext/S0092-8674(16)30909-6

Reporting on the 1st international training course on “Management and Operation of Imaging Core Facilities”, Global BioImaging H2020 Project November 16-18, EMBL, Heidelberg

20 participants attended this first course, representing European and international facilities (Argentina, Australia, India, South Africa). The course was organized around 4 sessions (Soft Skills for Core Facility Staff; Administrative Skills, Quality Management, Metrology; How to set-up an Imaging Core Facility (exchange workshop); E-learning/hands-on in virtual training platform) with a strong participation of the French Community members as speakers. All sessions were overall well received, as suggested by the feedback collected via survey. Some adjustments were however proposed that should frame the next GBI Training Course (planned in Australia, summer 2018). In the meantime, GBI will also organize the 2nd workshop “Exchange of Experience” for core facility managers, 15-16 September 2017 in Bangalore, India and will continue to develop its “shadowing” program (personal exchanges between imaging facilities worldwide; second session March  – September 2017). Stay tuned! For details, read more on the Euro BioImaging website.

France BioImaging was present at the ASCB 2016 meeting in San Francisco (December 3-7).  It was a great occasion to present our infrastructure on our booth and to draw future strategies in BioImaging for Cell and Development Biology.

We also interacted with scientific organizations and funders, such as the Howard Hughes Medical Institute,  the National Science Foundation and the Gordon and Betty Moore Foundation to whom we presented our R&D programs as well as the overall FBI organization. Most of them were amazed by what we could propose and surely other meetings and new programs could emerge from those informal although in depth discussions.

Beyond the above type of contacts, young scientists were attracted by FBI possibilities in terms of training capacity, accessibility to the most emerging technologies, software platform and lots of them were asking about FBI PhD and Post-doc programs. Certainly a prospect we could mine in the future.

As a general feedback on what were the active new fields of interest in the scientific area of the congress, let us mention the obvious developments in the Biology of Induced Pluripotent stem Cell (IPCs), CRISPR/Cas genome editing techniques, cell mechanics approaches and structure/function of macromolecular complexes in living cell and organisms. It is clear that FBI anticipation in developing super-resolution, optogenetics, multi-scale and highly sensitive imaging techniques will best serve our research teams focusing their interest in these diverse fields.

Finally, as many of our foreigner colleagues mentioned, attendance of French researchers was quite remarkable at ASCB this year. A number of them gave talks that were highly appreciated. A majority of them were coming from the direct perimeter of the FBI Nodes and clearly benefited from or even participated to the development of advanced technologies in imaging provided by France BioImaging.