The NeurImag cellular and molecular imaging Facility, member of the Paris Centre Node of France-BioImaging, has initiated the development of a new tool called ExoJ, in collaboration with the teams of Guillaume Van Niel (CRCI2NA, Nantes University), Frederik Verweij (Utrecht University), Thierry Galli (IPNP, Inserm, Université Paris Cité) and Junjun Liu (Shandong First Medical University).

What is ExoJ?

ExoJ is a plugin developed for the Fiji/ImageJ2 software, specifically designed to automate the reliable detection and analysis of exocytosis events from fluorescence microscopy images. Exocytosis is a cellular process where molecules or substances contained within a cell are released to the extracellular environment. This process involves the fusion of a vesicle, a membrane-bound sac, with the cell membrane. Once fused, the contents of the vesicle are expelled into the extracellular space.

How does ExoJ work?

ExoJ automatically identifies user-defined exocytosis events. It extracts key quantitative information such as the intensity, apparent size and duration of each event. ExoJ is fully parameterizable and configurable, making it suitable for studying different types of exocytosis, whatever the imaging modality (TIRF [1] and/or spinning disk [2]). ExoJ is a robust and reliable tool for analyzing large datasets!

What are the benefits of ExoJ?

ExoJ automates the detection of exocytosis events, considerably reducing analysis time compared with manual annotation. Moreover, the results obtained are reproducible, facilitating comparisons between different experiments. Finally, ExoJ is based on Fiji/ImageJ2, an open-source software widely used in the scientific community.

To read the article, click here.

[1] Cois et al., 2024 https://pubmed.ncbi.nlm.nih.gov/39145986/

[2] Hessvik et al., 2023 https://pubmed.ncbi.nlm.nih.gov/37285022/

Cellular junctions are essential to the integrity of epithelia, which cover most of our organs. In an article published in the journal PNAS, scientists, with among them members of our FBI Marseille node, reveal the existence of a new category of cell junctions. Using Stimulated emission depletion (STED) microscopy, they have put forward the need to reconsider the organization of intestinal cell junction described as such for more than 40 years.

Imaging intestine with STED

Stimulated emission depletion (STED) microscopy is a super-resolution technique that bypasses the diffraction limit of light microscopy to increase resolution. In our case, scientists were able to resolve the organization of complexes located at cell junctions with a resolution of a few tens of nanometers thanks to STED. Moreover, STED tripled the spatial resolution in the junctional plane and, using cryosections, they achieved imaging with a seven times greater spatial resolution compared to approaches that would use confocal microscopy and thus, without physical sectioning.

Although the resolution of STED is at least an order of magnitude lower than that of electron microscopy, the combination of STED with immunostaining reveals organization up to then unknown as multiple proteins can be efficiently labeled at the same time.

Three types of intestinal cell junctions

The intestine is covered with cells, most of which absorb the nutrients we ingest. These cells are joined together by three types of junctions which coexist and provide different functions, ranging from the selective filtration of certain ions to the mechanical maintenance of the epithelial layer. These junctions, the tight junction, the adherens junction, also called zonula adherens, and the desmosomes, were discovered in the 1960s and their constituent elements as well as their organization were proposed during the 1980s and 1990s.

The adherens junction in particular is established as being organized into a belt of adhesion proteins anchored to the membrane, the cadherins, and supported by filaments, the actin filaments. This junction has an important mechanical role in the cell, for example by impacting the shape of the cell. The zonula adherens (ZA), a fundamental module of epithelial cell–cell adhesion initially observed in intestinal cells, is believed to comprise a single contractile actin belt linked via E-cadherin-catenin to the ones of neighboring cells.

How did microscopy help reevaluate our current knowledge?

By observing the adherens junction of epithelial cells obtained from human intestinal biopsies, or from human cells in culture using STED super-resolution microscopy, scientists have made a very surprising discovery. They show that the ZA consists of two distinct belts of adhesive complexes, a basal one with E-cad-catenin and an apical one with nectin–afadin. Contrary to the prevailing view, the major actin belt aligns with nectin and afadin, not E-cad-catenin.

The authors further demonstrate that this organization depends on the cell maturation state and that the classical ZA found in textbooks corresponds to a less mature state of the intestinal junction. Therefore, they decided to call the junction found in mature cells the zonula adherens matura. Genetic and physical perturbations show that afadin is essential for force transmission across cell junctions. This work redefines the intestinal ZA architecture and prompts a reevaluation of how forces propagate within an epithelial sheet.

Not only, these results are important to better understand the adhesion and mechanics of epithelial cells, but these two essential characteristics of the epithelia are particularly affected in cancers of epithelial origin, which represent 80% to 90% of current cancers. This discovery is, thus, a step forward to the comprehension of cancers and to their treatment.

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You need FRAP, two photon FLIM-FRET, PALM/dSTORM at France-BioImaging? To get open access, please login via Euro-BioImaging website! You just have to choose the technology you want to use, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub in close relation with France-BioImaging. And of course, all scientists regardless of their affiliation, area of expertise or field of activity can benefit from open access services! Users whose projects will be validated by Euro-BioImaging will benefit from a waiver for the access cost on France-BioImaging core facilities (https://france-bioimaging.org/access/).

Fig.: Models of mature and immature intestinal cell junctions © Pierre Mangeol

Mangeol, P., Massey-Harroche, D., Sebbagh, M., Richard, F., Le Bivic, A., & Lenne, P. F. (2024). The zonula adherens matura redefines the apical junction of intestinal epithelia. Proceedings of the National Academy of Sciences, 121(9), e2316722121. https://doi.org/10.1073/pnas.2316722121

Sources : https://www.insb.cnrs.fr/fr/cnrsinfo/une-nouvelle-categorie-de-jonctions-cellulaires-dans-lintestin

https://www.ibdm.univ-amu.fr/redefining-epithelial-junctions-with-nanoscopy/

HIV type 1 virus has a lipid envelope enriched with host cell sphingomyelin and cholesterol. In order to understand the mechanism of this enrichment, the FBI Alsace node (Laboratoire de Bioimagerie et Pathologies from Université de Strasbourg and the Imaging Center PIQ-QuESt) has participated in a study recently published in Nature Communications about HIV-1 virus assembly. Indeed, they have investigated the interplay between the HIV-1 Gag protein and the host cell lipids at the plasma membrane. This work has greatly benefited from the use of a great combination of different quantitative (FLIM-FRET and FRAP) and super-resolution (PALM/STORM) custom-made microscopes with specific probes.

Using FRAP to characterize mobile and immobile molecules

The Fluorescence recovery after photobleaching (FRAP) quantifies the two-dimensional lateral diffusion of fluorescently labeled molecules of interest. This technique is very useful in biological studies of cell membrane diffusion and protein binding as it not only reports on the diffusion rates of mobile fractions of molecules but also provides information about the proportion of immobile molecules.

In our case, FRAP experiments indicated that the expression of Gag significantly decreased the mobile fraction of sphingomyelin (SM)-rich domains. Besides, the technique showed that cholesterol (Chol)-rich domains were intrinsically immobile, even in the absence of Gag. It is speculated that the association of Gag with SM-rich domains restricts the lateral diffusion of the lipid domains, resulting in an increase of the immobile fraction in FRAP measurements.

Using PALM/dSTORM to localize molecules at high resolution

The Photo-activated localization microscopy (PALM) is a widefield fluorescence microscopy imaging method that provides images with a resolution beyond the diffraction limit. By collecting a large number of images each containing just a few active isolated fluorophores, the collection of these images allows to stochastically activate each fluorophore and thus to obtain a global image of the sample with high resolution.

The Stochastic optical reconstruction microscopy (STORM) works on the activated state of a photo-switchable molecule that leads to the consecutive emission of sufficient photons to enable precise localization before it enters a dark state or becomes deactivated by photobleaching.

Coupling these two techniques, scientists next investigated at high resolution the localization of Gag and SM-rich or Chol-rich domains, both labeled with specific fluorescently labeled lipid binding proteins.

PALM/dSTORM visualized domains of different sizes labeled with the two lipid binding proteins, showing that the expression of Gag induced the formation of larger SM-rich domains but not the formation of larger Chol-rich domains. The main hypothesis is that the formation of large lipid domains may be due to the coalescence of smaller lipid domains.

Using FLIM-FRET to identify molecule proximity and interaction

And last but not least, the Fluorescence-lifetime imaging microscopy (FLIM) is an imaging technique for producing an image based on differences in the fluorescence-lifetime rather than its intensity. By quantifying variations in the exponential decay rate of the fluorescence from a fluorescent sample (fluorescence-lifetime) it is possible to report on molecule proximity. Since the fluorescence-lifetime is insensitive to changes in fluorophore intensity or concentration, it is the most quantitatively precise technique to report on fluorescence resonance energy transfer (FRET).

FRET is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through non-radiative dipole-dipole coupling. FRET is extremely sensitive to small changes in distance and therefore an excellent reporter on molecule proximity and interaction.

In this third and final part, to better understand the possible effect of Gag on the lipid distribution in the plasma membrane, scientists investigated by two-photon FLIM-FRET the interaction of Chol-rich lipid domains with SM-rich lipid domains and its dependence on Gag multimerization. These last results showed that Gag multimerization induces SM-rich and Chol-rich domains to be in close proximity and that membrane curvature affects the apposition of SM-rich and Chol-rich domains.

A great example of application on how a combination of high-end technologies in microscopy can help you understand multiple aspects of a biological mechanism. So, what are you waiting for? Dive into the true potential of microscopy!

Colocalization between Gag-mEos4b curvature mutants and AF647-NT-Lys and domain analysis in PALM/dSTORM (https://doi.org/10.1038/s41467-023-42994-w)

Get access to one of our services!

You need FRAP, two photon FLIM-FRET, PALM/dSTORM at France-BioImaging? To get open access, please login via Euro-BioImaging website! You just have to choose the technology you want to use, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub in close relation with France-BioImaging. And of course, all scientists regardless of their affiliation, area of expertise or field of activity can benefit from open access services! Users whose projects will be validated by Euro-BioImaging will benefit from a waiver for the access cost on France-BioImaging core facilities (https://france-bioimaging.org/access/).

Tomishige, N., Bin Nasim, M., Murate, M. et al. HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains. Nat Commun 14, 7353 (2023). https://doi.org/10.1038/s41467-023-42994-w

Technical information from www.eurobioimaging.eu

Most mammals can maintain a relatively constant and high body temperature. This is considered to be a key adaptation for theses species, enabling them to successfully colonize new habitats and survive harsher environments. Scientists from the Institut des Sciences de l’Evolution de Montpellier (ISEM) investigate the possible correlation between the maxilloturbinal in the anterior nasal cavity and the body temperature maintenance by using Micro Tomography (or MicroCT) at the MRI core facility (FBI Montpellier node). This technique was essential in this study as it rebuilt the hypothesis around body temperature maintenance. Here is what they found.

MicroCT: Image in a non-destructive way

First of all, what is Micro Tomography? Micro Tomography, or Micro-CT, is a 3D imaging technique using X-rays to see inside biological material, at a small animal or body part level. Slice by slice, this technology scans the object in a series of 2D images that are reconstructed in a 3D model. Micro CT is, thus, non-destructive. This means that it can be used to image a sample without having to cut it! Not only your material is still in one piece but you can use it for further experiments.

Phylogenetic studies as an example of application

In this study examining the correlation between skull structure and the stabilization of body temperature, MicroCT was the key. The presence and the relative size of the maxilloturbinal has been proposed as a hypothesis that reflects the endothermic conditions and basal metabolic rate in extinct vertebrates. Among bony structures, respiratory turbinals (e.g., maxilloturbinal) are interesting anatomical structures that may offer important insights to the origins of endothermy, in other words to the origin of warm-blooded animals. Indeed, respiratory turbinals are highly vascularized, which amplifies the surface area and offers an effective mechanism to avoid loss of internally-produced and costly heat.

You probably figured it out: scientists needed to compare the structure of the maxilloturbinal in order to take conclusion. This is when Micro Tomography was very useful. They scanned 424 individuals from 310 mammal species using high-resolution X-ray micro-computed tomography, with approximatively half of the samples imaged at MRI, part of our Montpellier node. Using the obtained comparative 3D µCT dataset, they explored the anatomical diversity of the maxilloturbinal based on relative surface area, morphology and complexity. They specifically tested the relationship between multiple parameters such as the size-corrected basal metabolic rate (cBMR), the relative surface area of the maxilloturbinal (Maxillo RSA) or body temperature.

And the results surprisingly showed that…

…there is no evidence to relate the origin of endothermy and the development of some turbinal bones! Even though scientists used a comprehensive dataset of Micro CT-derived maxilloturbinals spanning most mammalian orders, they demonstrate that neither corrected basal metabolic rate nor body temperature significantly correlate with the relative surface area of the maxilloturbinal. These results challenge the hypothesis of thermal regulation being linked to respiratory bone structure.

So, what could be linked with the thermoregulation of mammals? Researchers proposed 3 more hypothesis. First of all, environmental conditions could have a bigger role: “the maxilloturbinal function could have a more prominent heat/moisture exchange role in species that face harsh environmental conditions, thus helping to limit spurious heat and moisture loss”. Another major role of the maxilloturbinal is water conservation. As an example, the naked mole-rat avoid breathing through the mouth when performing energy intensive digging because the lips close behind the digging incisors and this species has the lowest value of predicted Maxillo RSA of the entire sample. But most of all, the factor could be a multifactorial physiological question. What is the relation of the maxilloturbinal with the overall nasal cavity? Do other functions play a role in the evolution of this body part, such as its protective role against toxic elements? Is it linked with brain cooling?

Well, imaging will certainly give them an answer in the future!

Detailed view of the maxilloturbinal in selected mammalian species with peculiar thermal and metabolic conditions or that undergo different forms of heterothermy (https://doi.org/10.1038/s41467-023-39994-1)

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You need Micro-CT or another imaging technology or expertise that France-BioImaging provides? To get open access, please login via Euro-BioImaging website! You just have to choose the technology you want to use, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub in close relation with France-BioImaging. And of course, all scientists regardless of their affiliation, area of expertise or field of activity can benefit from open access services! Users whose projects will be validated by Euro-BioImaging will benefit from a waiver for the access cost on France-BioImaging core facilities (https://france-bioimaging.org/access/).

Martinez, Q., Okrouhlík, J., Šumbera, R. et al. Mammalian maxilloturbinal evolution does not reflect thermal biology. Nat Commun 14, 4425 (2023). https://doi.org/10.1038/s41467-023-39994-1

A key property of the human cornea is to maintain its curvature and consequently its refraction capability. Although we know that it is related to its stacked collagen lamellae structure, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. A team from the Laboratory for Optics and Biosciences (LOB) has optimized a recent technology which combines Second Harmonic Generation microscopy and polarimetry (P-SHG) to image the lamellar microstructure of human corneas and more!

Acquire the structure in depth

Imaging the cornea is essential to understand how visual acuity works. This part of the eye is characterized by its transparency and refractive power but also by its unique mechanical properties. To answer the many questions of the cornea structure, the Second Harmonic Generation microscopy is the perfect technique. This technology is based on the sample capacity to generate second harmonic light, which has half the wavelength of the light entering the material. However, the SHG is working on well-aligned assemblies of non-centrosymmetric molecules which fits perfectly with the collagen!

Apart from being specific to this kind of macromolecules, the SHG microscopy offers multiple advantages. First of all, as it does not involve the excitation of molecules, molecules do not suffer from phototoxicity or photobleaching effects. Moreover, no markers are necessary which makes this type of microscopy noninvasive. Finally, Second Harmonic Generation microscopy allows the visualization of in-depth structure of thick samples. As a matter of fact, it is, nowadays, the gold standard technique for in situ visualization of collagen 3D organization in unstained biological tissues.

Add polarimetry and get orientation information

P-SHG first offers all the advantages of usual SHG microscopy: 3D optical imaging in depth and high specificity and sensitivity to collagen without any labeling. In this study, scientists took advantage of the light polarization to reveal the direction of the collagen fibrils that make up the lamellae of the cornea in their SHG microscopy acquisition. This recent technology is called: polarization-resolved SHG microscopy (P-SHG). The main novelty of this study was to implement P-SHG in depth to analyze intact human corneas along their full thickness (up to 600 µm). 3D reconstructions of P-SHG data show in a unique way the stacking of collagen lamellae with different orientations all along the thickness of the cornea. Here, imaging helped confirm that these lamellae are roughly organized parallel to the cornea surface, with different collagen orientations in sequential lamellae, and provided new information about the variation of these orientations along the depth of the cornea.

Aside from being the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution, this imaging technique could be a huge step forward in vivo diagnosis as it uses the detection of the reflected signal. Furthermore, this study opens the way to promising new characterizations of the cornea, such as mapping the size and distribution of lamellae as a function of depth, but also as a function of position (center or periphery of the tissue). This information will feed into mechanical modelling of corneal behavior during variations in intraocular pressure or healing processes. Finally, the study of pathological tissues will clarify the role of the corneal defective structure in certain diseases.

These results show the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues. And of course, this approach is readily applicable to more than just cornea! It may be used for instance to decipher the structure of collagen in fibrotic pathologies or in other proteins that exhibit SHG, namely myosin and tubulin, or in starch and cellulose in plants. This shows the unique potential of P-SHG microscopy for imaging thick collagen-rich tissues.

Three-dimensional reconstruction of the lamellae structure of a human cornea. Colors indicate the direction of the collagen lamellae in the imaging plane, as shown in the inset color wheel. The image size is 250 x 250 x 600 µm3. The anterior part (side outside the eye) of the cornea is at the top of the image.

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Polarization SHG microscopy is not available in open access but we are open to collaborations!

You need classic SHG microscopy or another imaging technology or expertise that France-BioImaging provides? To get open access, please login via Euro-BioImaging website! You just have to choose the technology you want to use, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub in close relation with France-BioImaging. And of course, all scientists regardless of their affiliation, area of expertise or field of activity can benefit from open access services! Users whose projects will be validated by Euro-BioImaging will benefit from a waiver for the access cost on France-BioImaging core facilities (https://france-bioimaging.org/access/).

Sources: https://www.inp.cnrs.fr/fr/cnrsinfo/cartographier-la-structure-de-la-cornee-humaine

https://www.ip-paris.fr/en/news/unveiling-structure-human-cornea

Raoux, C., Chessel, A., Mahou, P. et al. Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy. Light Sci Appl 12, 190 (2023). https://doi.org/10.1038/s41377-023-01224-0

 

In 2022, in a project funded by the Euro-BioImaging pilot User Access Fund, Andrew Boyce, postdoctoral fellow at the University of Calgary, used state-of-the-art microscopy techniques at the Bordeaux Imaging Center, part of the Bordeaux node of France-BioImaging, for panoptical visualization of brain tissue in vivo. His work just got published in a Nature Communications article!

The advantages that shadow imaging has in visualizing brain tissue

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Unfortunately, current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy).

In this study, scientists have shown how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide the information lacking in the previous techniques. Basically, it’s like looking at the negative of a photo! Called Shadow Imaging, they have demonstrated the power of this approach on revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts.

The perfect combination between the right technology and the right expertise

How did Andrew Boyce contribute to this study? Interested in blebbing neuron dendrites during cell death caused by a stroke, he only had, at that time, results from how cells in culture reacted during this kind of events but no information about the neighboring cells.

Thanks to Euro-BioImaging pilot User Access Fund, he came to Bordeaux to use a combination of live cell 3D stimulated emission depletion (3D-STED) microscopy and super-resolution shadow imaging (SUSHI), and adapt shadow imaging approaches to conventional confocal microscopy (COSHI). Shadow imaging allows you to visualize fine details of cell-cell interactions and the extracellular space in an unbiased manner. Brain tissue is complex and shadow imaging is a technique that allows researchers to visualize all of the cells in this very complex architecture in an unbiased manner so it was the perfect match for Andrew’s research project!

A career-boosting experience

“This has really been an incredible experience. It’s amazing to be trained by the person and team who pioneered this technique. I’m very thankful for this opportunity and humbled to be over here,”reflects Andrew from Bordeaux.

When asked how his visit to Bordeaux might impact his career, Andrew explains that he can certainly imagine doing shadow imaging in the future. He would eventually like to run his own lab at an academic institution in Canada. Being able to bring an exciting and novel technique like this back to Canada would be really impactful for his career, and for science in general.

“Shadow imaging is a very exciting technique for studying brain tissue, but getting started on sample preparation and adapting this technique to your research question can be daunting. Coming to the Bordeaux Imaging Center to work with the person who developed this approach and get expert training is a dream come true and will make a big difference to my research. This is an amazing opportunity made possible by the Euro-BioImaging Pilot User Access fund with the goal of making shadow imaging more accessible across diverse platforms,” concludes Andrew.

Andrew Boyce during his stay
at the Bordeaux Imaging Center (www.eurobioimaging.eu/news/using-super-resolution-live-cell-imaging-to-understand-cell-death-during-stroke)

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You want to be the next user to get access to state-of-the-art technologies that France-BioImaging provides? Please login via Euro-BioImaging website! You just have to choose the technology you want to use, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub in close relation with France-BioImaging. And of course, all scientists regardless of their affiliation, area of expertise or field of activity can benefit from open access services! Users whose projects will be validated by Euro-BioImaging will benefit from a waiver for the access cost on France-BioImaging core facilities (france-bioimaging.org/access)

Thanks to Marianna Childress and Andrew Boyce for the original article!

Dembitskaya, Y., Boyce, A.K.J., Idziak, A. et al. Shadow imaging for panoptical visualization of brain tissue in vivo. Nat Commun 14, 6411 (2023). https://doi.org/10.1038/s41467-023-42055-2

Microglial navigation through complex brain tissue is revealed by confocal shadow imaging (https://doi.org/10.1038/s41467-023-42055-2)

Atomic Force Microscopy (AFM) is a scanning probe microscopy technique that relies on measuring the interaction forces between a sharp tip and the surface of a sample to generate high-resolution images of its surface features and mechanical properties. A very broad range of sample types can be imaged with this technique at a very high resolution – at sub-nanometer level for some of them! Discover the AFM at the Montpellier node of France-BioImaging with Christine Doucet from Integrative Biophysics of Membranes team of the Centre de Biochimie Structurale.

Quickly visualize dynamic biological processes with High-Speed AFM

AFM provides images in physiological conditions, in liquid, over a length-scale ranging from few nanometers (single biomolecules) to tens of micrometers (living cells). In fact, the resolution depends on the tip radius and sample properties. For some of them, you can routinely obtain a nanometer lateral resolution and Angstrom axial resolution!

You want a video-rate version of the biological samples you are imaging? The High-Speed AFM, permits the acquisition of movies at approximately 10 images per second, enabling the visualization at nanoscale of dynamic biological processes involving biomolecular interactions, diffusion or conformational changes. It delivers nanometric resolved images typically at the same speed as conventional fluorescence microscopes!

Unravel the chemical information of your sample by combining AFM with…

AFM in ambient conditions and in liquids has a key limitation in that it does not directly provide chemical information about the sample being imaged. However, this limitation can be overcome by combining AFM with other techniques to obtain additional information about the sample’s composition. 

One commonly used technique in correlation with AFM is fluorescence microscopy. This combined approach of fluorescence labeling and AFM provides valuable insights into the chemical and biological properties of the sample. It was recently used on the Montpellier custom-made correlative AFM / fluorescence setup to observe the sublocalization of proteins in HIV-1 budding sites 1. They also used it to unambiguously attribute some unexpected configurations of the nucleoplasmic sides of Nuclear Pore Complexes 2. In these two cases, fluorescently-labeled proteins were imaged by dSTORM (direct STochastic Optical Reconstruction Microscopy). Of note, the lateral resolution of dSTORM and AFM are both in the 20 nm range with such samples, which makes their combination ideal!

In addition to fluorescence microscopy, AFM can also be correlated with other complementary techniques to obtain chemical information about the sample, such as Raman spectroscopy, Infrared Spectroscopy, X-Ray spectroscopy, microscopy and scattering.

Learn more about AFM applications

Here are 2 studies where Atomic Force Microscopy were essential: 

  • Structure and mechanics of the human nuclear pore complex basket using correlative AFM-fluorescence superresolution microscopy

Combining mechanical and superresolution measurements to reveal the plasticity of the Nuclear Pore Complexes

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells, facilitating the transport of selected cargoes of size from a few up to hundred nanometers. This versatility implies an important pore plasticity. Here, by combining atomic force microscopy (AFM) and single molecule localization microscopy (SMLM), a group led by France-BioImaging R&D team members Christine Doucet and Pierre Emmanuel Milhiet revealed that the NPC basket is very soft and explores a large conformational landscape: apart from its canonical basket shape, it dives into the central pore channel or opens, highlighting  how this structure can adapt, and let morphologically diverse cargoes shuttle through NPCs.

Vial et al., Nanoscale, 15, 5756-5770 (2023) 

  • The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity

Structural Biology meets Correlative Imaging

Huntington’s disease is a neurodegenerative disorder caused by an extended polyglutamine (poly-Q) tract in huntingtin. Here, using NMR, the team of Pau Bernado (CBS Montpellier) demonstrated that this poly-Q tract adopts long α-helical conformations. By adding correlative Atomic Force Microscopy and Fluorescence Microscopy data obtained in the FranceBioImaging facility PIBBS in Montpellier, they could demonstrate that the stability of this α-helix is a stronger signature than the number of glutamines, in defining the aggregation kinetics and the structure of the resulting fibrils, potentially linked to their pathogenicity.

Elena Real et al., Nature Structural & Molecular Biology, 30, 309–320 (2023)

How to use Atomic Force Microscopy at France-BioImaging?

Atomic Force Microscopy is open to collaborations under Proof-of-concept studies via Euro-BioImaging webportal (www.eurobioimaging.eu/service)! At the Montpellier node of France-BioImaging, you will be in contact with Dr Luca Costa (costa@cbs.cnrs.fr) with whom you will talk about the feasibility and the inherent experimental constraints linked to the technique. The collaboration procedure is discussed on a case-by-case basis, depending on the duration and technicity of the required experiments. Feel free to submit your project!

Thanks to Christine Doucet and Emmanuel Margeat for providing helpful information!

1. Dahmane, S., Doucet, C., Le Gall, A., Chamontin, C., Dosset, P., Murcy, F., Fernandez, L., Salas, D., Rubinstein, E., Mougel, M., et al. (2019). Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM. Nanoscale 11, 6036–6044.

2. Vial, A., Costa, L., Dosset, P., Rosso, P., Boutières, G., Faklaris, O., Haschke, H., Milhiet, P.-E., and Doucet, C.M. (2023). Structure and mechanics of the human nuclear pore complex basket using correlative AFM-fluorescence superresolution microscopy. Nanoscale 15, 5756–5770.

Age-related macular degeneration (AMD) affects more than 150 million people worldwide (early AMD) and 10 million of patients suffer from debilitating late stage AMD. Blurring central vision, this eye disease progresses over time, usually beginning when people are around their 50s or 60s by causing damage to the macula, in the retina. Researchers from the Institut de la Vision (Sorbonne Université, INSERM, CNRS, UMR_S 968) recently published about the AMD. Thanks to Serial Block-Face Scanning Electron Microscopy (SBF-SEM) experiments carried out at the ImagoSeine core facility (Institut Jacques Monod / FBI Paris-Centre node), they describe in this new study melanophages as a disease-progression marker.

Early or intermediate AMD is characterized by pigmentary changes and lipoproteinaceous debris accumulation between the photoreceptors and the melanosome-rich retinal pigment epithelium (RPE) or below the RPE. Later, AMD can be complicated by central choroidal neovascularization or by an expanding lesion of the photoreceptors. Even though patients with early or intermediate AMD can progress and develop late AMD, a large part of patients stay stable for years, underlining the potential usefulness of progress. 

AMD is associated with the appearance of hyperreflective foci, with reflectivity comparable to melanocyte-containing RPE cells. Thbs1 and CD47 are both important for the elimination of these cells. In the absence of either of them, melanocyte-containing RPE cells would then accumulate. The goal was to determine the origin of these cells in the retina, and the main question was: are these cells RPE migrating to the wrong place, or melanosome phagocytes cells having ingested melanosomes?

SBF-SEM: the key to answer this question

The Serial Block-Face Scanning Electron Microscopy (SBF-SEM) is a 3D electron microscopy imaging technique, where an ultramicrotome is placed inside a SEM. Biological samples are beforehand stained with heavy metals and embedded in a plastic resin block. Inside the microscope, a thin-section is cut at the surface of the block and discarded. Then, an image of the surface of the block – therefore inside the sample – is made, using back-scattered electrons. The process of cutting and imaging is repeated automatically as many times as necessary to produce a 3D stack of images inside the sample, as it is progressively imaged and destroyed. 

This technique allows 3D imaging of large samples for Electron Microscopy standards (up to several hundred microns in each of the X,Y,Z direction) at high resolution. This technique is often used to image whole cells, or even small pieces of tissues in 3D. The two major domains of application are to:

  • find a rare structure within a cell or tissue. The sample is imaged until the structure of interest is found.
  • understand the 3D spatial organization of organelles within cells, or of cells between them.

The benefits of bioimaging in this study

In the study, SBF-SEM was essential. As previously mentioned, AMD is associated with the appearance of hyperreflective foci, with reflectivity comparable to melanocyte-containing RPE cells. In the images produced by SBF-SEM, the retinal pigment epithelium (RPE) surrounding the melanophages in mice, where CD47 was inhibited, were markedly less pigmented and deformed compared to those where Thbs1 was blocked. This suggests that melanosomes have been transferred by phagocytosis from the RPE to nearby melanophages because they lack CD47. Finally, authors have shown that CD47 acts as a “don’t eat me” signal. The SBF-SEM was a great addition to this study where understanding the 3D spatial organization of the structure of interest was key.

Thanks to Jean-Marc Verbavatz for providing very helpful insights of the study!

Augustin, S., Lam, M., Lavalette, S. et al. Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker. J Neuroinflammation 20, 28 (2023). https://doi.org/10.1186/s12974-023-02699-9

Get access to one of our services!

You need SBF-SEM or another imaging technology or expertise that France-BioImaging provides? To get open access, please login via Euro-BioImaging website! You just have to choose the technology you want to use, then submit your proposal. All applications will be processed by the Euro-BioImaging Hub in close relation with France-BioImaging. And of course, all scientists regardless of their affiliation, area of expertise or field of activity can benefit from open access services! Users whose projects will be validated by Euro-BioImaging will benefit from a waiver for the access cost on France-BioImaging core facilities (https://france-bioimaging.org/access/)

Massive intracellular accumulation of RPE-derived melanosomes in subretinal MPs of CD47−/−-mice causes subretinal melanophage formation and their clinical appearance as hyperreflective foci.

Brettanomyces bruxellensis is one of the most damaging spoilage yeasts in the wine industry because of its impact on the beverage’s flavor. Lysiane Brocard, research engineer specialised in plant biology at the Bordeaux Imaging Center (FBI Bordeaux node), recently co-published an article on this yeast cell surface and bioadhesion properties.  

Fruits are transformed into beverages through fermentation processes carried out by microorganisms naturally present in the environment. In wine, yeasts and bacteria play this role and contribute to the development of volatile compounds. Scientists targeted Brettanomyces bruxellensis in this study, a yeast famous for the production of volatile phenols, characterized by horse sweat odors which is – usually – not very enjoyable for the consumer. 

A yeast characterized by bioadhesion abilities

This specific odor comes from volatile compounds, the 4-ethylgaïacol (4EG) and 4-ethylphenol (4 EP), that winemakers try to avoid. Beside adding an unpleasable flavor to the beverage, the issue is that the spoilage yeast is persistent in cellars over several years, resulting in recurrent wine contamination. This suggests a bioadhesion process that helps the microorganism to survive in its environment. To put it simply, bioadhesion is the ability of an organism to adhere on a surface to, then, participate in the formation of a biofilm (which is defined as “a structured community of microorganisms adhered to a surface and producing an extracellular matrix”). 

Here, 54 strains of B. bruxellensis were characterized for their cell surface physico-chemical and bioadhesion properties. And all of them have shown bioadhesion abilities (after only three hours on stainless steel) both on synthetic medium and wine. Enough to highlight the persistence of our favorite horse sweat flavored yeast. 

How did bioimaging help in this project?

Among all the analytical methods used in this study, microscopy helped identify the structure of the biofilms formed with B. bruxellensis. Two imaging techniques were used: confocal microscopy and scanning electron microscopy. The first one offers the advantage of realizing live imaging without being too time-consuming. With fluorescent dyes, the status of cells can be easily determined at the same time as the cell repartitions and concentrations.

Moreover, comparisons have been made thanks to confocal microscopy to determine if some strains of B. bruxellensis could form biofilm with only one cell layer or if they proliferate in three dimensions. To complete these observations, scanning electron microscopy was performed at the Bordeaux Imaging Center (FBI Bordeaux node) with the help of Isabelle Svahn, expert of this type of microscopy. It was a great addition to this study as these observations validated the morphological variability among Brettanomyces strains.

Bioimaging helped a broader project about Brettanomyces led by Isabelle Masneuf-Pomarède who works in the Institut des Sciences de la Vigne et du Vin of Bordeaux. Isabelle studies the persistence and proliferation of Brettanomyces over the years. Isabelle’s PhD student, Paul Le Montagner, carried out most of the experiments published in this paper. Thanks to them for this amazing paper!

Confocal microscopy observations after 3h of bioadhesion of cells on stainless steel
SEM observation of 3h-aged cells adhered on stainless steel

Original article: Paul Le Montagner, Morgan Guilbaud, Cécile Miot-Sertier, Lysiane Brocard, Warren Albertin, Patricia Ballestra, Marguerite Dols-Lafargue, Vincent Renouf, Virginie Moine, Marie-Noëlle Bellon-Fontaine, Isabelle Masneuf-Pomarède, High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis, Food Microbiology, Volume 112, 2023, 104217, ISSN 0740-0020, https://doi.org/10.1016/j.fm.2023.104217

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Researchers from the Laboratory for Optics and Biosciences (LOB, CNRS / École Polytechnique / INSERM), a member of the Ile-de-France Sud node of France-BioImaging, and from the Developmental Biology and Stem Cells Department (UMR3738, CNRS / Institut Pasteur), developed a new form of multiphoton microscopy providing label-free imaging of red blood cells and oxygenation. This technique is called color third-order sum-frequency generation microscopy, and is described in an article recently published in Light: Science & Applications.

Keeping resolution, saving time

Current methods used to map microcirculation and blood oxygenation at high resolution typically require the injection of fluorescent or phosphorescent markers. In addition, they usually require relatively long pixel times and thus are limited in spatio-temporal resolution. The novel approach is based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. In practice, this microscopy is based on the illumination of samples by two pulsed infrared lasers and the simultaneous detection of several TSFG and THG signals emitted at different colors. This method has the advantage of providing simultaneous measurements at several wavelengths spanning the hemoglobin absorption spectrum. This simultaneity makes TSFG microscopy appropriate for studying dynamic samples…

To better understand brain and tissue physiology

…such as biological tissues! Biological tissues are supplied with oxygen by red blood cells, responsible for circulating hemoglobin through the body. Scientists have shown that the intensity of the different signals detected by TSFG microscopy depends on their spectral proximity to the absorption wavelength of hemoglobin. As the color of hemoglobin depends on its oxygenation state, the TSFG makes it possible to image red blood cells circulating in live zebrafish larvae – and even to probe their oxygenation state. Researchers also demonstrated that this contrast modality is also compatible with deep-tissue microscopy and can be used to observe the brain of a live adult zebrafish. An example that confirms the broad range of application of this novel imaging technique.

(a) TSFG color microscopy principles ; (b) Imaging of isolated red blood cells ;
(c) Imaging of a zebrafish embryo ; (d) 3D in vivo imaging in an adult zebrafish brain.
© Laboratoire d’optique et biosciences (LOB, CNRS / École Polytechnique / INSERM)

Source: https://www.inp.cnrs.fr/fr/cnrsinfo/imager-haute-resolution-les-globules-rouges-et-loxygenation-dans-les-tissus-biologiques

Reference: Ferrer Ortas, J., Mahou, P., Escot, S. et al. Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy. Light Sci Appl 12, 29 (2023). https://doi.org/10.1038/s41377-022-01064-4

Published on August 23rd, 2022 in EMBO reports, this article questions the way that core facilities should be recognized in the scientific literature and their key contributions to data lifecycle. An initiative endorsed by France-BioImaging.

Core facilities are an integral part of the life science research landscape as providers of centralised access to technological resources and expertise. This article’s working group has estimated that between 40 and 80% of imaging, proteomics and genomics data at their institutes are generated at core facilities. The contribution of core facilities to scientific research and innovation must thus be accordingly recognised. In that respect, the most straightforward way is an acknowledgement. Unfortunately, the lack of formal rules still leaves core facilities being inadequately recognised. 

This article proposes that the recognition of core facilities should be deployed via two actions and implemented in two phases: first, with the systematic acknowledgement of core facilities in all scientific publications, and second, by including core facilities and their staff in data citations (Cousijn et al, 2018).

The first step can be accomplished at the manuscript-submission stage by asking the corresponding author to confirm if any data (and associated metadata) used in the manuscript originated from a core facility, and if yes, to identify the associated core facility. EMBO Press has recently included a question in the author checklist to confirm whether the work in the publication “benefited from core facilities” and that the core facility be acknowledged accordingly.

The next step would be to make it compulsory for authors to respond to such a query and explicitly identify the core facility and relevant data (and associated metadata). The MDAR (Materials, Design, Analysis, Reporting) form (Macleod et al, 2021), wherein one needs to provide information about data availability in the Analysis section, could likewise include a question to explicitly identify core facilities involved. Eventually, the information in the author checklist could be automatically fed into the acknowledgement section.

Acknowledging will have two key positive consequences: on the sustainability of core facilities and on their staff careers. In the absence of a high number of publications, particularly as lead or corresponding authors, acknowledgements are used as a measure of a core facility and its staff’s output and impact. Second, it further motivates and incentivizes core facility staff to actively contribute to scientific research. 

The acknowledgement of a core facility goes beyond professional courtesy: identifying the origin of data (and associated metadata) is essential for data traceability and reproducibility particularly since core facilities are major generators of data in life science research. 

Thanks to Jean SALAMERO, our “Action inter-infrastructures” mission officer, for contributing to this article. 

Full article on:

Acknowledging and citing core facilities

Katja Kivinen, Henri G A M van Luenen, Myriam Alcalay, Christoph Bock, Joanna Dodzian, Katerina Hoskova, Danielle Hoyle, Ondrej Hradil, Sofie Kjellerup Christensen, Bernhard Korn, Theodoros Kosteas, Mònica Morales, Krzysztof Skowronek, Vasiliki Theodorou, Geert Van Minnebruggen, Jean Salamero, Lavanya Premvardhan

EMBO reports (2022) 23: e55734
https://doi.org/10.15252/embr.202255734

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