Mercredi 07 juillet
09:40

Mercredi 07 juillet

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SC2
09:40 - 12:15

Symposium Commun 2
Méthodes d’imagerie volumique (SBEM, AT, FIB-SEM)

Modérateurs : Nicolas BROUILLY (IBD, Marseille), Williams LEFEBVRE (GPM, Rouen)
Les contributions concerneront des travaux menés par toute technique d’imagerie tridimensionnelle opérant depuis le micromètre jusqu’à l’échelle atomique et utilisée pour résoudre des problématiques liées au domaine du vivant ou des matériaux inorganiques. L’accent sera mis sur les cartographies tridimensionnelles de propriétés, l’imagerie 3D résolue en temps, la corrélation de données tridimensionnelles, l’analyse d’échantillons à plusieurs échelles au moyen de protocoles expérimentaux parfois périlleux et souvent originaux et toutes les approches émergentes dans le domaine de l’imagerie volumique.
09:40 - 10:10 The 3D approach in electron holography. Christophe GATEL (CEMES, Toulouse)
L’holographie électronique est une technique interférométrique permettant d’enregistrer le déphasage de l’onde ayant traversé l’échantillon. Ce déphasage permet d’obtenir des cartographies quantitatives à l’échelle du nanomètre des potentiels/champs électriques et magnétiques et d’accéder ainsi aux propriétés électromagnétiques locales du nanosystème étudié. Cependant ce déphasage provient d’une intégration le long du trajet électronique des potentiels et l’image finale obtenue n’est que la projection bidimensionnelle de configurations électromagnétiques tridimensionnelles. Il faut ainsi tenir compte des variations d’épaisseur, des champs rayonnés, d’artefacts de préparation et de l’effet de charge du faisceau électronique afin d’interpréter pleinement les résultats obtenus. Dans cet exposé, je montrerai les paramètres dont il faut tenir compte et les approches que nous avons développées pour reconstituer les configurations électromagnétiques tridimensionnelles des systèmes étudiés. Ces approches permettent à l’holographie électronique d’être considérée directement ou indirectement comme une méthode d’imagerie volumique.
10:10 - 10:20 Discussion.
10:20 - 10:50 Les techniques basées sur le MEB pour l’acquisition de larges volumes en science de la vie. Christel GENOUD (PME UNIL, Lausanne, Suisse)
Les MEB sont utilisées désormais de manière routinière pour acquérir des images sériées d’échantillons biologiques. Grâce au FIBSEM, de petits volumes sont acquis à haute resolutions. Trois exemples vont illustrer les apports de cette technique pour explorer des échantillons au niveau cellulaire. Le microtome-SEM permet de très grandes et longues acquisitions. L’exemple d’un organisme complet acquis et de la corrélation avec des données génétiques illustrera cette technique. Finalement, l’essor de l’”array tomography”, technique non-destructive, permet de détecter des événements rares et de combiner l’analyse 3D ultrastructurelle avec des immunomarquages.
10:50 - 11:00 Discussion.
11:00 - 11:15 #26228 - Imagerie FIB-SEM et reconstructions 3D pour étudier le phytoplancton.
Imagerie FIB-SEM et reconstructions 3D pour étudier le phytoplancton.

Phytoplankton encompasses a huge diversity of eukaryotic unicellular organisms, issued by multiple endosymbiosis events, and belonging to distinct taxa. Their heterogeneous origin has led to a large biodiversity of phytoplankton, which comprises cells with different morphologies and sizes. Although they are exposed to extremely variable environments, phytoplankton cells are on average fast growing, suggesting that their metabolism should be highly flexible. However, we still do not know if these original metabolic strategies are linked to singular cell topological arrangements, due to the lack of high resolution imaging studies. 

We have developed a FIB-SEM–based workflow (Fig. 1) to generate 3D reconstructions of different eukaryotic microalgae representing major oceanic phytoplankton lineages suitable for quantitative morphometric analysis (surfaces and volumes) of organelles and subcellular structures. 

Cells were cryo-fixed using high-pressure freezing followed by slow freeze substitution and resin embedding. FIB-SEM datasets were processed to 3D models using open-access software. We obtained complete 3D models of entire cells including quantification of cellular and subcellular volumes, surfaces and interactions between compartments (Fig. 2).

By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales [1]. 

[1] Uwizeye, C., al. (2021) Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging. Nat Commun 12, 1049. https://doi.org/10.1038/s41467-021-21314-0.


Clarisse UWIZEYE (Grenoble)
11:15 - 11:25 Discussion.
11:25 - 11:40 #26231 - Analyse quantitative 3D de la structure et de la morphologie de la boehmite hiérarchique.
Analyse quantitative 3D de la structure et de la morphologie de la boehmite hiérarchique.

Boehmite (AlOOH) is considered as an important precursor to γ-Al2O3 which when calcinated undergoes topotactic transformation to form the latter.1,2 Alumina has extensive applications in fields such as catalysis, abrasives, cosmetics and many more. Boehmite falls under the category of hierarchical structures whose structural and textural properties are a result of its compositional and porous hierarchy. Although research has been carried out exquisitely to understand the complete representation of its structure, a true morphological model is an important key to understand and fully explain its transport properties during catalytic processes.3,4

3D electron microscopy in TEM or STEM modes helps us to dive deeper into the different hierarchical entities of boehmite, bridging the gaps between the models and assumptions made using the traditional characterisation techniques such as N2 Physisorption, Hg Porosimetery, X-Ray Diffraction (XRD), Dynamic Light Scattering (DLS) and Small angle X-Ray Scattering Techniques (SAXS). We present here deeper insight into the structural and morphological parameters of different commercial boehmites (Disperal 40, Dispal 10F4, Catapal 200 and Pural TH500) using 3D-TEM. The extraction of quantitative descriptors of the hierarchical entities and their comparison to bulk analyses provide a truthful and detailed insight into such microstructures. We show how seamingly identical grades of boehmite differ by their varying homogeneity of entity sizes and more particularly by different platelet interaction configurations giving rise to different pore types (Figure.1). The interaction between the different crystallographic planes - (200), (020) and (002) is found to be different from one sample to another giving rise to the uniqueness in its different compositions within its aggregates and the combination of four different morphologies quantified was different from aggregates of one boehmite grade to another (Figure.2).

References:
[1] Christoph, G. G. Clays Clay Miner. 27, 81–86 (1979).
[2] Wilson, S. J. J. Solid State Chem. 30, 247–255 (1979).
[3] Karouia, F. Powder Technol. 237, 602–609 (2013).
[4] Wang. H. J. of Microscopy. 260, pp. 287–301(2015).


Nivedita SUDHEER (Strasbourg)
11:40 - 11:50 Discussion.
11:50 - 12:05 #26232 - Méthode de reconstruction 3D de cellules par STEM en phase liquide vers l'étude des interactions cellule-biomatériaux.
Méthode de reconstruction 3D de cellules par STEM en phase liquide vers l'étude des interactions cellule-biomatériaux.

The successful development of new implants relies on a better understanding of the interactions between cells and biomaterials [1]. Cell adhesion onto the biomaterial is a key step to predict the future of an implant in the human body. Cell adhesion is involved in many cellular processes such as proliferation, migration and differentiation. Recent studies have shown that the substrate on which cells are cultured can lead to different cell morphologies, which is linked to the expression of specific genes [2]. Cell adhesion is widely studied through focal adhesion labelling in confocal microscopy. The morphological analysis is mostly done in 2D using high vacuum scanning electron microscopy, a label-free technique requiring heavy sample preparation including dehydration. To reduce changes during sample preparation, a solution may be the use of liquid-phase SEM (LPSEM) analysis. A home-made scanning transmission electron microscopy (STEM) tomography device, specific to LPSEM, has previously been developed to study aqueous suspensions in 3D [3]. In this work, we further develop the device and propose a 3D analysis method to study whole cells in 3D close to their native state. We will show that Liquid-Phase electron tomography in ESEM enables the 3D analysis of whole hydrated cells without any heavy sample preparation. We will first present the technical settings used for image acquisition on hydrated biological objects. Then, we will show complementary volumes reconstructed from BF and HAADF tilt image series (see figure 1 A and B). Morphological parameters describing cell parts, such as the filopodia, will be extracted. The limits of the technique will be explored in terms of culture medium, cell thickness, spatial resolution and electron dose received by the sample. Complementary topographic results of cells/thick substrates will also be analysed. Such development opens the way towards 3D analysis of intact cells in environments mimicking their native environment. Another targeted application is the 3D study of the morphological interactions between cells and biomaterials.

[1] Cells Mater., 3, (1993) p.141–150.
[2] Nat. Commun., 11, (2020) p 1–13.
[3] Microsc. Microanal., 20, (2014) p.366–375.


Akkiz BEKEL (Lyon)
12:05 - 12:15 Discussion.
Room 1
12:10 Pause
13:10

Mercredi 07 juillet

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SDV2
13:10 - 15:45

Symposium Sciences de la Vie 2
Cryométhodes 2 : Cellulaires / Tissulaires : applications et développements

Modérateurs : Amélie LEFORESTIER (Chargée de Recherche) (Paris-Saclay), Patrick SCHULTZ (IGBMC, Strasbourg)
Les développements récents des cryo méthodes cellulaires et tissulaires offrent la possibilité de décrire leur organisation au niveau moléculaire. Un ensemble de technologies à basse température doit être combiné pour préserver au mieux l’ultrastructure cellulaire par cryo fixation, amincir la cellule par cryo-coupe ou fabrication de cryo-lamelles, reconstruire le volume cellulaire par cryo tomographie électronique et déterminer la nature, la structure et les interactions des molécules présentes dans ce volume. L’opportunité de visualiser des molécules en action dans leur contexte cellulaire sans avoir à les purifier et donc les séparer de leurs partenaires est une révolution majeure dans le domaine de la biologie. De nombreuses difficultés techniques restent à surmonter pour démocratiser ces approches, reconnaitre sans ambiguïtés les machines moléculaires de petite taille ou peu abondantes, ou encore améliorer la résolution des reconstructions tridimensionnelles. L’ambition de cette session est de présenter l’état de l’art et d’illustrer les développements par des applications biologiques représentatives des progrès les plus récents.
13:10 - 13:40 Towards a mechanistic understanding of ciliary assembly by CLEM and cryo-ET. Gaia PIGINO (MPI-CBG, Dresden, Allemagne)
Research in my lab aims at understanding the molecular principles and processes that drive the self-organization of complex cellular machines, which are relevant for human health and disease. In this talk I will show how we combine EM and molecular cell biology methods to obtain a mechanistic understanding of the molecular machineries required for the assembly and the function of cilia/flagella: conserved organelles that are fundamental for most eukaryotic cells.
Assembly of the cilium requires the rapid bidirectional intraflagellar transport (IFT) of building blocks to and from the site of assembly at its tip. This bidirectional transport of IFT trains is driven by the anterograde motor kinesin-2 and the retrograde motor dynein-1b, which are both bound to a large complex of about 25 IFT adaptor proteins. We developed a millisecond resolution 3D correlative light and electron microscopy (CLEM) approach to show that the spatial segregation of oppositely directed IFT trains on the two microtubules of each axonemal doublet ensures a collision free transport in the cilium. Then, it remained to be explained how competition between kinesin and dynein motors, both found on anterograde IFT trains, is avoided. In other bidirectional transport systems in the cell the presence of opposing motors leads to periodic stalling and slowing of cargos moving along the microtubule. No such effect occurs in IFT. To address this question, we used cryo-electron tomography and sub-tomogram averaging to resolve the 3D structure of IFT train complexes in the cilia/flagella of Chlamydomonas cells. We showed that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an inhibited conformation and by positioning it away from the microtubule track to prevent binding. We also show that dyneins are released from the train at the ciliary tip, but how this happens and how these motors are activated to power retrograde IFT is not yet understood. Protein complexes that form specialized structures at the ciliary tip are thought to be involved in this process. By mechanically manipulating IFT trains in the cilia/flagella of Chlamydomonas cells, we showed that the structures of the ciliary tip are not necessary for the conversion from anterograde to retrograde IFT. Thus, the conversion process is an intrinsic, calcium-optional ability of IFT machinery. Other research lines of the Pigino Lab aim at understanding the role of tubulin posttranslational modification for the assembly and the mechanics of cilia and flagella, and the structure, composition and function of primary cilia.
13:40 - 13:50 Discussion.
13:50 - 14:20 Structural biology of prokaryotic cell surface proteins. Tanmay BHARAT (Bacterial Biofilms, Oxford Univ., Royaume-Uni)
My laboratory studies how molecules on the surface of prokaryotic cells mediate cellular interaction with the environment, enabling cellular motility, initiating cellular adhesion to surfaces, and facilitating biofilm formation. For our work, we leverage our expertise in electron cryotomography (cryo-ET) in situ imaging, together with ongoing method development in subtomogram averaging approaches for structure determination of macromolecules in their native context. We combine cryo-EM with FIB milling of specimens and cryo-light microscopy to study molecules on prokaryotic cells.
14:20 - 14:30 Discussion.
14:30 - 14:45 #26368 - HEMNMA-3D: a new method for continuous conformational variability analysis of macromolecules. Application to cryo electron tomography of nucleosomes in situ.
HEMNMA-3D: a new method for continuous conformational variability analysis of macromolecules. Application to cryo electron tomography of nucleosomes in situ.

Cryogenic electron tomography (Cryo-ET) allows in situ structural determination of macromolecules. Its potential to provide information on macromolecular dynamics is still largely unexploited due to data analysis challenges represented by 1) low signal-to-noise ratio and 2) spatial anisotropies due to the limited tilt angle of Cryo-ET (missing-wedge).

Conventional subtomogram analysis techniques simplify macromolecular conformational variability to discrete rather than continuous solutions using 3D-subtomogram classification and class averaging.

We present HEMNMA-3D [1], the first method for analyzing subtomograms for macromolecular continuous conformational changes. HEMNMA-3D uses elastic and rigid-body alignments of a flexible 3D-reference (atomic structure or a density map) to match the macromolecule's conformation, orientation, and position in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal-Mode-Analysis of the reference) and subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge. The conformational parameters (amplitudes of normal modes) of the macromolecules in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into macromolecular dynamics, by calculating 3D-averages of subtomograms with similar conformations from selected (densest) regions and recording movies of the 3D-reference's displacement along selected trajectories through the densest regions.

In this presentation, we describe HEMNMA-3D and show its application on an experimental dataset describing in situ nucleosomes.

HEMNMA-3D software is available freely as part of the ContinuousFlex plugin of Scipion-V3.

[1] Harastani M, Eltsov M, Leforestier A, Jonic S. HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes. Front. Mol. Biosci, in press.

Acknowledgments:

We acknowledge the support of the French National Research Agency—ANR (ANR-20-CE11-0020-01 to A.L., ANR-20-CE11-0020-02 to M.E., ANR-20-CE11-0020-03 and ANR-19-CE11-0008-01 to S.J.); German Research Foundation (DFG EL 861/1 to M.E.); La Société Française des Microscopies (2019 Master internship grant to M.H.); the Sorbonne University (2019 ""Interface pour le Vivant"" PhD scholarship grant to M.H.); and the access to the HPC resources of CINES and IDRIS granted by GENCI (2019-A0070710998, AP010712190, AD011012188 to S.J.).


Mohamad HARASTANI (Paris)
14:45 - 14:55 Discussion.
14:55 - 15:10 #26357 - Microtubule lattice organization in Xenopus laevis egg cytoplasmic extract.
Microtubule lattice organization in Xenopus laevis egg cytoplasmic extract.

Our current knowledge on microtubule structure is essentially derived from the numerous studies that have been performed on microtubules assembled in vitro from purified tubulin. However, little is known concerning the structure of microtubules in cells, and more specifically on the organization of the tubulin heterodimers within their lattice. 

To tackle this matter, we have used Xenopus egg cytoplasmic extracts that can recapitulate the events of the cell cycle [1], and thus constitute a convenient close-to-cellular open system where proteins can be either added or depleted. Here, we used the motor domain of kinesin that binds every tubulin dimer to decorate microtubules in the egg cytoplasm. Dual-axis cryo-electron tomographic data were taken so that all microtubules could be analyzed irrespective of their orientation with respect to the tilt axes [2] (Fig. 1). We then used a segmented sub-tomogram averaging approach to analyze the organization of the tubulin molecules within all microtubules (Fig. 2).

We found that the vast majority of the microtubules are made of 13 protofilaments with 3-start lateral helices. Tubulin molecules engage homotypic lateral interactions of the B-type along the 3-start helices, except at a unique region of the A-type, called the seam. Yet, we observed exceptions such as microtubules segments built from different protofilaments number (e.g. 12 and 14), as well as fully helical 13 protofilaments microtubule segments with 4-start lateral helices. We also found that the seam location can vary within individual microtubules, which implies the presence of holes inside their wall. In parallel, we have analyzed the structure of microtubules assembled in vitro from pure tubulin and discovered that they are much more structurally heterogeneous, both in terms of protofilament and seam numbers, as well as in lattice type transition frequency (~40 times higher in vitro than in Xenopus egg cytoplasm). Altogether, our data suggest that microtubule architecture is tightly regulated in cells to avoid the formation of lattice defects commonly observed in microtubules assembled in vitro from purified tubulin. 

References:

[1] Gibeaux R. and R. Heald. Cold Spring Harb. Protoc. (2019), doi:10.1101/pdb.top097048 

[2] Guesdon A. et al. J. Struct. Biol., 181 (2013), p. 169–78.


Charlotte GUYOMAR (Rennes)
15:10 - 15:20 Discussion.
15:20 - 15:35 #26359 - Sparse cryo-STEM tomography for biological samples.
Sparse cryo-STEM tomography for biological samples.

Cryo-electron tomography (Cryo-ET) enables to recover 3D information of cryo-fixed samples in a near-native state [1] with a thickness up to ~250 nm [2]. Thicker samples (up to 1 µm) can be studied in scanning transmission electron microscopy (STEM) [3][4]. At equivalent electron dose, STEM induces less radiation damage to the sample than classical transmission electron microscopy (TEM) [5] and is able to image micrometer thick cryo-fixed biological samples in 3D (Fig. 1) [6]. However, in order to image thicker specimens or to reach higher magnifications we need to increase the dwell time or to sample the specimen more densely, respectively. This will inevitably increase the electron dose and the beam damage proportionally. The need to further reduce the electron dose emerges. 

In this work, we present a sparse cryo-STET acquisition scheme. Instead of collecting a full frame for each tilt-angle, we collect only a fraction of the pixels. This permits to reduce the electron dose received by the sample on each frame and across the full tilt-series. Multiple sparse data collection schemes have been previously investigated at our lab [7] (Fig. 2). A pre-processing inpainting step, using discrete cosine transform (DCT) [8] or shearlets transform [9], is necessary prior tilt-series alignment to recover the missing information due to the data sparsity. Then the 3D volume can be reconstructed using classical methods like WBP [10] or iterative algorithms such as SIRT [11]. 

To demonstrate the advantages of a sparse cryo-STET acquisition scheme, we applied our method on a sample that is too thick (>1µm) to be studied using conventional TEM methods.

References 

[1] Lučić et al., J. Cell Biol., 202(2013), pp.407–419.

[2] Aoyama et al., Ultramicroscopy, 109(2008), pp.70–80.

[3] Midgley & Weyland, Ultramicroscopy, 96(2003), pp.413–431.

[4] Pennycook, Ultramicroscopy, 123(2012), pp.28–37.

[5] Wolf et al., Nat. Methods, 11(2014), pp.423–428.

[6] Trépout, J. Struct. Biol. X, 4(2020), p.100033.

[7] Trépout, Materials, 12(2019), p.2281.

[8] Garcia, Stat. Data Anal., 54(2010), pp.1167–1178.

[9] Kutyniok et al., GAMM-Mitteilungen, 37(2014), pp.259–280.

[10] Radermacher, in Electron Tomography, Springer, (2006), pp.245–273.

[11] Gilbert, J. Theor. Biol., 36(1972), pp.105–117.


Antoine COSSA (Paris-Saclay)
15:35 - 15:45 Discussion.
Room 1

Mercredi 07 juillet

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SDM3
13:10 - 15:45

Symposium Sciences de la Matière 3
Cristallographie, structures et microstructures

Modérateurs : Emmanuel BOUZY (LEM3, Metz), Christine LEROUX (IM2NP, Toulon)
La cristallographie est à la base de l'élaboration de tous les nouveaux matériaux. En effet, afin d'optimiser et de développer de nouvelles fonctionnalités, l'arrangement atomique, les orientations, les déformations élastiques des cristaux et les défauts qu’ils présentent doivent être étudiés dans l'espace réel et/ou dans l'espace réciproque, et ce à différentes échelles. Pour ce faire, que ce soit en MEB ou en MET, différentes techniques de diffraction et d'imageries sont utilisées parmi lesquelles : EBSD, TKD, ECCI, HRTEM, ACOM, PED, NBED, CBED, STEM in SEM, STEM in TEM… Ce symposium se veut général et regroupe les études de structure et microstructure de matériaux sous forme massive, de poudres ou de couches minces, qui font appel aux techniques ci-dessus ou à toutes autres techniques relatives à la cristallographie. Les contributions qui établissent un lien certain entre l'étude structurale et microstructurale et les propriétés du matériau sont les bienvenues.
13:10 - 13:40 ANALYSE MICROSTRUCTURALE DES SUPERALLIAGE BASE NICKEL POLYCRISTALLINS POUR DISQUES DE TURBINE. Nathalie BOZZOLO (CEMEF, Paris)
Les superalliages polycristallins sont utilisés pour la fabrication des disques de turbine de motorisation aéronautique. Les alliages employés aujourd’hui et pour les quelques décennies à venir sont de type γ-γ’. La microstructure des disques forgés doit répondre à un cahier des charges strict pour atteindre les propriétés requises pour cette application parmi les plus exigeantes. Aussi le développement des gammes de fabrication suppose la parfaite connaissance des mécanismes et cinétiques métallurgiques à l’œuvre au cours des opérations de forgeage à chaud.
Ainsi l’optimisation de la tenue en service de ces alliages passe par l’analyse fine des microstructures en cours de mise en forme et par l’analyse des mécanismes de recristallisation qui contrôlent la taille de grains finale. La cartographie d’orientations par EBSD est une technique phare dans ce domaine.
Les superalliages γ-γ’ présentent des difficultés particulières liées notamment à la similitude cristallographique de la matrice austénitique et de la phase secondaire de structure L12 et au caractère multi-échelle des populations de précipités. Différentes approches possibles pour contourner ces difficultés et obtenir des cartographies d’orientation discriminant les deux phases seront présentées (variantes de la technique EBSD et techniques alternatives) [1,2].
Par ailleurs l’analyse quantitative des microstructures visant à l’évaluation de l’énergie stockée, pilote de la recristallisation, sera également illustrée et discutée [3,4].

Références:
[1] Charpagne M.-A., Vennéguès P., Billot T., Franchet J.M. & Bozzolo N., Journal of Microscopy, 263:1 (2016) p.106-112
[2] Vernier S., Franchet J.M., Lesne M., Douillard T., Silvent J., Langlois C. & Bozzolo N., " Materials Characterization, 142 (2018) p. 492-503
[3] Seret A., Moussa C., Bernacki M., Signorelli J. & Bozzolo N., Journal of Applied Crystallography, 52 (2019) p.548-563
[4] Nicolay A., Franchet J.M., Cormier J., Mansour H., De Graef M., Seret A. & Bozzolo N., " Journal of Microscopy, 273:Issue 2 (2019) p.135-147
13:40 - 13:50 Discussion.
13:50 - 14:20 Measuring local lattice parameters by Precession Electron Diffraction: an opportunity to give a personal overview of Electron Crystallography. Jean-Luc ROUVIÈRE (Docteur) (LEMMA, Grenoble)
In this presentation, I will start recalling what is crystallography and explain why some of my work - in particular strain measurement by electron diffraction with or without precession (Fig. 1) - can be related to crystallography. After recalling this strain measurement work, I will show some appealing recent publications on electron crystallography and will take this opportunity to give a non-crystallographer and thus partial overview of the evolution of Electron Crystallography.

Figure 1 gives an overview of local strain measurements by N-PED (Nano-beam Precession Electron Diffraction). Diffraction Maps (DM), also called 4D-STEM experiments, are performed by scanning in 2 directions an electron beam of about 1 nm in diameter, and at each position a diffraction pattern (DP) is recorded and numerically analysed. Using simple filtering and autocorrelation, the relative local lattice parameters can be measured accurately; by using a reference pattern either locally in the same map or in a second map obtained on a reference sample of known lattice parameter absolute measurements can be obtained with an accuracy approaching 10-4, and a spatial resolution of about 1 nm [1]. When the sample contains only homethetic diffraction patterns, like in a SiGe/Ge multilayer (Fig. 1a), the strain formula is straightforward. When the sample contained different phases like in a GaN layer deposited on Si [3] (Fig. 1b), the strain formula involves several matrix multiplications.
This method has been applied to study strain in different semiconductor nanostructures [1-8] but it could be applied in any crystalline nomaterial, where 2D-projected strain maps bring useful information on the structure.

Références/References :
[1] J.-L. Rouviere et al. Appl. Phys. Lett., vol. 103, no 24, (2013)
[2] S. Reboh et al., Appl. Phys. Lett., 112 (2018) p. 051901
[3] N. Mante et al., Journal of Applied Physics, 123 (2018) p. 215701
[4] T. Cerba et al. Thin Solid Films, vol. 645 (2018) p. 5 9
[5] S.-T. Zhang et al., Materials & Design, 132 (2017) p. 518
[6] B. Haas et al., Appl. Phys. Lett. 110 (juin 2017) p. 263102,
[7] D. Cooper et al., Appl. Phys. Lett. 110 (2017) p. 223109
[8] J. Aubin et al., Semiconductor Science and technology 32, no 9, (2017)
14:20 - 14:30 Discussion.
14:30 - 14:45 #26401 - An original homography-based approach for high-angular resolution EBSD and TKD: principle and examples.
An original homography-based approach for high-angular resolution EBSD and TKD: principle and examples.

Digital image correlation (DIC) techniques usually performed on deforming speckle patterns are transferred to electron microscopy in order to map grain internal disorientation with high angular resolution, as well as geometrically necessary dislocation densities (GND) and elastic strains.The developed method analyses electron diffraction patterns as a whole through a unique and large region of interest for which the relative deformations are described by a linear homography [1]. Such a geometric transformation is often met in photogrammetry to model projections. It is here measured by means of a numerically efficient inverse-compositional Gauss-Newton algorithm (IC-GN). The latter integrates a correction of optical distortions caused by the camera lenses, avoiding thus a time-consuming patterns straightening step [2]. A global cross-correlation based initial guess step pre-aligns the  patterns so that the IC-GN algorithm convergences despite the presence of disorientations up to 10°. The method is implemented in ATEX-software [3], developed at our lab, and applied to a semi-conductor and a deformed metal. First, the elastic strain field in the vicinity of a screw dislocation in GaN single crystal is investigated using EBSD. It reproduces very well the theoretical elastic strain field (Fig. 1). Then, the DIC analysis is coupled with the recent on-axis Transmission Kikuchi Diffraction (TKD) configuration to characterise a nanocrystalline aluminium obtained by severe plastic deformation. The dislocation structures are highlighted by the GND density maps (Fig.2). Thanks to this coupling, high-spatial (3-6 nm) and high-angular (0.01°) resolutions are simultaneously achieved in the scanning electron microscope [4].

References:

[1] C. Ernoud et al., Acta Materialia 191 (2020), p.131-148

[2] C. Ernoud et al., Ultramicroscopy, 221 (2021), p.113158

[3] B. Beausir, J.-J. Fundengerger, Analysis Tools for Electron and X-Ray Diffraction, ATEX - Software, Université de Lorraine, Metz, 2017.

[4] C. Ernoud et al., Scripta Materialia, 185 (2020), p.30-35

Figure 1: (a) Forward Scatter Detector (FSD) image of a screw dislocation in GaN and (b-d) elastic strain maps obtained by the HR-EBSD method.

Figure 2: On-axis HR-TKD characterization of a nanocrystalline aluminium and comparison with a standard indexation-based analysis.


Clément ERNOULD (Metz)
14:45 - 14:55 Discussion.
14:55 - 15:10 #26412 - Low-dose imaging of metal-organic frameworks and study of their electron-beam stability.
Low-dose imaging of metal-organic frameworks and study of their electron-beam stability.

Metal-organic frameworks (MOFs) are hybrid (organic/inorganic) crystalline porous materials intensively studied for their potential in several applications related to energy, environment or health. Diffraction-based techniques are the methods of choice for the crystal structure determination of MOFs, providing a better understanding of their unique properties. However, the information obtained is the averaged periodic structure, while in some cases - such as adsorption and catalysis - the local structural features (ie., crystal surfaces, interfaces, the presence of guest molecules or defects) are key elements. In this regard, high-resolution TEM methods provide the opportunity to probe/visualize such details [1]. However, MOFs are among the most beam-sensitive materials and can be easily damaged after exposure to few electrons/Å2. Therefore, there are only a few studies where HRTEM imaging was successful, so far. Most of them focus on a few MOFs that were found to be relatively stable under the electron beam [1]. These recent studies have demonstrated that using a direct detection electron counting camera (DDEC) allows performing HRTEM imaging of MOFs with extremely low dose rates (as low as 4 e−/Å2.s) [2].

The prediction of the electron-beam stability of a MOF nanocrystal is a difficult task since many structural, morphological or instrumental parameters can be taken into account and could have interconnected effects. This work aims at identifying these parameters and assessing their influence on the stability of these objects under the beam. Several known MOFs were successfully imaged by low-dose HRTEM (Figure 1) enabling the investigation of the effect of the voltage, the particle size and orientation, the presence of guest molecules, as well as the nature of the organic and the inorganic moieties. Additionally, low-dose HRTEM imaging has been for the first time applied on new in-house nano-sized MOFs (Figure 2), where important structural information was extracted and confirmed by complementary techniques [1].

Acknowledgement :

The authors acknowledge the Region Ile-de-France in the framework of DIM ResPore for funding.

Références/References :

[1] L. Liu, D. Zhang, et al., Communications Chemistry (2020), 3, 1–14.

[2] Y. Zhu, J. Ciston, et al., Nature materials (2017), 16, 532–536.


Asma MANSOURI (Paris)
15:10 - 15:20 Discussion.
15:20 - 15:35 #26413 - Combined Electron Diffraction and Chemical Contrast Imaging Studies : Identification of Nanometric Inclusions in Annealed (Eu,Tb) Doped ZnO/Si Junction.
Combined Electron Diffraction and Chemical Contrast Imaging Studies : Identification of Nanometric Inclusions in Annealed (Eu,Tb) Doped ZnO/Si Junction.

(Tb,Eu) doped ZnO films annealed at 1100°C showed intense photoluminescense (PL) emission from Eu and Tb ions [1]. The high temperature annealing led to a chemical segregation and a secondary Zn free phase formation that is suspected to be responsible for the high PL intensity. Large faceted inclusions of rare earth silicates of a size of few hundred nanometers were observed (Figure 1). Owing to various advanced electron microscopy techniques, a detailed microstructural study of these nanometric inclusions combining atomic Z contrast imaging (STEM) and precession electron diffraction tomography (PEDT) data was carried out and concluded to the formation of a hexagonal P63/m-type (Tb,Eu)9.43(SiO4)6O2-δ structure related to an oxyapatite structure [2]. Simulated atomic imaging and chemical analyses from spectroscopic data (EDX mapping and EELS) at the atomic scale (Figure 2)  have confirmed this result and showed that both rare earth (RE) elements sitting on two independent atomic sites have three-fold oxidation states while refinements of their occupancy sites from PEDT data have evidenced preferential deficiency for the first one. The deduced RE-O distances and their corresponding bond valences are discussed with the efficient energy transfer from Tb3+ towards Eu3+.

References :

[1] Davesnne, C.; Ziani, A.; Labbé, C.; Marie, P.; Frilay, C.; Portier, X. Energy Transfer Mechanism between Terbium and Europium Ions in Zinc Oxide and Zinc Silicates Thin Films. Thin Solid Films 2014, 553, 33–37.

[2] Leroux, Chris; Guillaume, Clement; Labbe, Christophe; Portier, Xavier and Pelloquin, Denis. Identification of (Tb,Eu)9.43(SiO4)6O2-delta Oxy-Apatite Structures as Nanometric Inclusions in Annealed (Eu,Tb)-Doped ZnO/Si Junctions: Combined Electron Diffraction and Chemical Contrast Imaging Studies. Inorganic chemistry 2021, 60,7, ‏  4508-4516 


Denis PELLOQUIN (Caen)
15:35 - 15:45 Discussion.
Room 2
15:40 Pause
16:00

Mercredi 07 juillet

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POS1
16:00 - 18:00

Session Posters 1

16:00 - 18:00 #26224 - STEM-in-SEM FOR LOW COST STRUCTURAL BIOLOGICAL STUDIES AT ROOM TEMPERATURE.
STEM-in-SEM FOR LOW COST STRUCTURAL BIOLOGICAL STUDIES AT ROOM TEMPERATURE.

Marie Laure SGARRA *, Aurelie DELIOTFranck CHARLESSergio MARCO

  • Sanofi Pasteur, Analytical Sciences, 1541 avenue Marcel Merieux, 69280 Marcy l’Etoile.
  • JEOL Europe SAS. 1 Allée de Giverny, 78290 Croissy sur Seine.


Ultrastructural studies of proteins, viral particles and resin embedded cells by negative staining are often performed by transmission electron microscopy (TEM). TEMs, in general, require a significant investment and maintenance cost having limited possibilities to program an automatic data acquisition flow from several grids inserted at once. STEM-in-SEM microscopes [1] are low cost solution compared to TEMs for acquisition, operation and maintenance. It offers the advantage of a multi-EM-grids holders (up-to 12 TEM-standard 3mm-grids) automatic management becoming a valuable alternative to address the aforementioned issues. In addition, they are easier to use not requiring additional protocols for sample preparation for negative staining or resin-embedding samples such as they are equal to those used for TEM. We aim here to demonstrate how STEM-in-SEM can be an alternative to make it available for a maximum number of laboratories. All the biological structural studies which can be performed at room temperature from cells to viruses and proteins including 2D-single-particle analysis (2D-SPA) which would be the maximum analytical limit of this technique as it requires the maximum achievable resolution. We have compared two studies with the same ~650 kDa oligomer performed by SPA using the same grids by MET and STEM-in-SEM. Raw images examples shown in figure 1 depict the ability of STEM-in-SEM to generate data suitable for 2D-SPA. We will introduce a full comparative study performed on these images’ sets. In addition, we will illustrate the use of STEM-in-SEM of large resin embedding samples study, without heavy metals post-embedding contrasting, at high magnification by stitching. In this last case, no charging phenomena were observed with a good enough contrast to get access to subcellular structures. In summary, STEM-in-SEM allows the negatively stained samples study, being even suitable for 2D single particle analysis, as well as for ultrastructural studies of large areas of resin embedded samples. STEM-in-SEM becomes a powerful and economic alternative to TEM for ultrastructural biological studies at room temperature. References: [1] Woolf et al., A transmission stage for the scanning electron microscope, J. Phys. E, 5 (1972), p. 230-23
Marie Laure SGARRA (Sanofi Pasteur)
16:00 - 18:00 #26225 - Real-time drift correction for EELS hyperspectrum acquisition.
Real-time drift correction for EELS hyperspectrum acquisition.

Marcel Tencé *, Xiaoyan LiAlexandre GloterMichael Walls

  • LPS Orsay (CNRS)


We present a novel approach for drift correction in real time during EELS hyperspectrum (HS) acquisition, taking advantage of the high-speed and noiseless readout characteristics of newly available direct electron detectors. These enable several HS to be recorded in sequence while adjusting the scanned area between each HS to remain on the same area of the sample. To achieve this, the cross-correlations between successive HAADF images (acquired simultaneously with the HS) are used to estimate the drift occurring during each scan. With our Medipix3RX Merlin EM detector, nominal dwell times as low as 100µs are currently attainable if one-bit counting is used. For quantitative, linear counting this limits the counts per channel per scan to somewhat less than the number of channels over which the spectrum signal is spread in the non-dispersive direction (about 150 in our case). But in the core-loss region such low count rates are typical using sub-millisecond dwell times. This approach eliminates the need for a separately defined drift-calculation image as is commonly used at present but which often presents difficulties, since a suitable region is not always available. Figure 1 shows an HAADF image and elemental maps of the of the Sc L (ca. 400eV) and Dy M (ca. 1300eV) signals from a conventional HS acquisition without drift correction on an ScDyO3 crystal oriented along the [100] pseudo-cubic axis. The dwell time was 20ms, giving a total acquisition time of 444s. Considerable drift occurred, producing distortions in the images. Figure 2 shows the equivalent results using our approach. Here, 100 HS each with a nominal pixel dwell time of 200µs were collected from the same area and summed. The total signal acquisition time is thus the same as in Figure 1 and the count levels were similar. However, the resulting image and maps are essentially free of drift distortion. We believe this method is the way forward for EELS HS acquisition. We thank Quantum Detectors for implementing TCP streaming speed improvements in the Merlin software, and ESTEEM3 (Europe), METSA (France) and the “Equipex” project TEMPOS (France) for financial support.
Marcel TENCÉ (ORSAY)
16:00 - 18:00 #26226 - Orientation mapping of bio-inspired alumina down to 1 kV.
Orientation mapping of bio-inspired alumina down to 1 kV.

C. LafondT. DouillardH. SaadS. DevilleS. MeilleP. SteyerS. CazottesC. Langlois *

  • Univ Lyon, INSA Lyon, UCBL, CNRS, MATEIS, UMR5510, 69621 Villeurbanne, France
  • Laboratoire de Synthèse & Fonctionnalisation des Céramiques, UMR3080 CNRS-Saint-Gobain CREE, Saint-Gobain Research Provence, Cavaillon 84306, France
  • Univ Lyon, Université Claude Bernard Lyon 1, CNRS, Institut Lumière Matière, 69622 Villeurbanne, France


Mapping the grain orientations of polycrystals brings crucial information when studying the relationships between microstructure and properties. Among different orientation mapping approaches, Electron Back Scattered Diffraction (EBSD) in a Scanning Electron Microscope (SEM) has been a milestone in the development of crystallographic characterizations. Nevertheless, efforts are still needed to improve the technique toward a better spatial resolution. One strategy is to decrease the accelerating voltage in order to reduce the interaction volume under the probed surface. However, the Kikuchi pattern quality decreases rapidly and the standard indexation routine fails. The actual limit found in the literature is 5kV [1]. Orientation mapping using the eCHORD approach [2] is not subjected to such limitations: it has been shown that the channeling of back scattered electrons (BSE), was still present at low voltages. In the current work, we show for the first time that orientation mapping at 1 kV is possible. The test material was a bio-inspired alumina presenting a lamellar structure mimicking the shell nacre, developed to improve the resistance to crack propagation. EBSD and eCHORD results about the local crystallographic texture are in very good agreement. This preliminary result paves the way for addressing samples sometime difficult to map by conventional EBSD like deformed crystal, nanocrystalline grains or subgrains, and insulating materials. Acknowledgments Thanks are due to the CLYM (www.clym.fr) for access to the ZEISS NVision 40 scanning electron microscope. C. Lafond thanks the French Ministry of Higher Education and Research for his PhD grant. We are also grateful for the financial support from the Agence Nationale pour la Recherche (ANR-16-CE08-0006 BICUIT, program DS0303-2016). References: [1] Singh, S., Guo, Y., Winiarski, B., Burnett, T. L., Withers, P. J., & De Graef, M. (2018). High resolution low kV EBSD of heavily deformed and nanocrystalline Aluminium by dictionary-based indexing. Scientific reports, 8 (2018), 10991 [2] Lafond C., Douillard T., Cazottes S., Steyer P. and Langlois C. (2018) Electron CHanneling ORientation Determination (eCHORD): An original approach to crystalline orientation mapping. Ultramicroscopy, 186 (2018), p.146-149
Clément LAFOND ()
16:00 - 18:00 #26227 - Characterisation of PAN and PES polymeric filtration membranes for microalgae valorization using 3D FIB/SEM.
Characterisation of PAN and PES polymeric filtration membranes for microalgae valorization using 3D FIB/SEM.

Hélène Roberge *, Philippe MoreauEstelle CouallierPatricia Abellan

  • Institut des Matériaux Jean Rouxel de Nantes
  • Laboratoire de Génie des Procédés, Environnement et Agroalimentaire, GEPEA, Saint Nazaire


Membrane filtration processes allow concentrating, separating and purifying the components from a complex mixture in a liquid phase. Recently, they have been adapted for microalgae valorization, where filtration employing nanoporous polymer membranes is used to separate and recover lipids and proteins from ground microalgae aqueous extracts. The biomolecules can be used in pharmaceutical industry, cosmetics, food supplements or biofuel industry [1]. The performance of the membrane is related to the size and density of its pores, but also to the selective layer thickness (a thin layer placed in contact with the fluid to be filtered), as shown by Poiseuille’s law [2]. During filtration, the unwanted accumulation of biomolecules at the surface and in the pores, termed fouling, hampers membrane performances. A detailed characterization of the pore structure, firstly in clean membrane, is essential to understand the fouling. 3DFIB/SEM allows characterizing the 3D structure of the material with a few nanometers resolution. Data acquisition is particularly challenging with polymer membranes constituted of amorphous materials presenting little contrast and a strong sensitivity to the electron and ion beams. Moreover, 3D reconstructions of porous media present a common issue: the shine through artefact, (material from subsequent slices is imaged through the pores), leading to a pores deformation in the FIB-milling direction [3]. We tested two widely used polymer membranes: PAN (polyacrylonitrile) and PES (polyethersulfone), with a respectively nominal pore size of their selective layer of 30 nm and 100 nm. We developed a 3D FIB/SEM method to obtain quality 3D reconstructions with a 5 nm voxel size and overcoming beam sensitivity and artefact difficulties. In this presentation, the sample preparation as well as the optimization of acquisition and reconstruction parameters will be described. We will present a method for identifying the selective layer in polymer filtration membranes using FIB/SEM. The quantitative analysis of pore size distribution, porosity, connectivity and tortuosity (figure 1) will be discussed. The properties-structure relationships will be analyzed, by linking these results with the theory of Hagen-Poiseuille on the porous media flow calculation. References: [1] Rivera et al., PB (2020) [2] AIMAR et al., TI, (2010) [3] Terao et al., JPS 347 (2017)
Hélène ROBERGE (Nantes, Saint Nazaire)
16:00 - 18:00 #26234 - 3D Microstructure Study of Li1.5Al0.5Ge1.5(PO4)3 Solid electrolyte pellets using FIB/SEM Tomography.
3D Microstructure Study of Li1.5Al0.5Ge1.5(PO4)3 Solid electrolyte pellets using FIB/SEM Tomography.

Sorina Cretu *, Linh Lan NguyenVincent SeznecArash JamaliLoic DupontArnaud DemortièreMartial Duchamp

  • Laboratoire de Réactivité et Chimie des Solides, UMR CNRS 7314, 33 Rue Saint Leu, 80039 Amiens, France
  • Réseau sur le Stockage Electrochimique de l’Energie (RS2E), FR CNRS 3459, 80039 Amiens, France
  • School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Technological University, Singapore 639798, Singapore


Inorganic solid electrolytes (SE)[1] represent an advantageous future technology due to their non-flammable nature, good stability with high voltage cathode materials and extended temperature opration ranges. Li1.5Al0.5Ge1.5(PO4)3 (LAGP) [2], oxide SE gained attention due to its wide electrochemical window, high moisture stability and high ionic conductivity which can be obtained by sintering them at high temperature. The total conductivity of LAGP pellet has an inverse relationship with the amount of grain boundaries of the inside electrolyte pellet since Li+ ions are conducted relatively slower at at the grain boundaries due to solid-solid interface. SE pellets with different densities were prepared by Spark Plasma Sintering (SPS) and investigated using techniques such as Potentiostat Electrochemical Impedance Spectroscopy (PEIS), Focused Ion Beam/ Scanning Electron Microscopy (FIB/SEM) tomography and SEM-Energy Dispersive X-rays (EDX) analysis in order to get a better understanding better of the link between the amount of grain boundaries and ionic conductivity. FIB/SEM tomography analysis (Fig.1a) using a Dual Crossbeam 540 Zeiss microscope offered insights into the 3D morphology of solid electrolyte pellets and revealed unexpected presence of an extra phase at LAGP grain boundaries (Fig.1c). The back-scattered SEM images were segmented using a Random Forest approach with WEKA FIJI plugin allowing us to identify each phases (LAGP, extra-phase and porosity). The quantitative image process of 3D volume allowed us to extract porosity distribution, tortuosity and phase-to-phase surface area. Tomographic data displayed a lower presence of the grain boundary phase for high compact pellets indicating a clear correlation between the extra phase and the total ionic conductivity, which is consistent with PEIS analysis. SEM-EDX analysis showed that the extra phase located at the grain boundaries contained less germanium compared to the pure LAGP SSE. [1] Zhang, Z. et al. Energy Environ. Sci. 11, 1945–1976 (2018). [2] Delaizir, G. et al.. Adv. Funct. Mater. 22, 2140–2147 (2012).
Sorina CRETU (Amiens)
16:00 - 18:00 #26229 - FIB-SEM to study the Intraflagellar transport.
FIB-SEM to study the Intraflagellar transport.

Adeline Mallet *

  • Institut Pasteur


Cilia and flagella are essential organelles composed in general of 9 doublet microtubules. They can adopt different configurations from one species to the other, or even from one cell type to the other in the same organism. Their construction is mainly done by addition of new subunits at the distal end. Precursors are transported by Intraflagellar transport (IFT), the movement of trains composed of protein complexes between the flagellar membrane and the microtubule doublets driven by molecular kinesin and dynein motors. Despite high train frequency in both anterograde and retrograde directions (1-3 trains per second), collisions are extremely rare. We proposed three hypotheses to explain this phenomenon based on differential localisation for anterograde and retrograde trains. The localisation of IFT trains within flagellum was not precisely determined so far. We studied the localisation of IFT trains in the flagellum of the protist Trypanosoma brucei. Thanks to FIB-SEM, a three-dimensional electron microscopy approach, we established that IFT trains are localised specifically on only 4 microtubule doublets out of the 9 available. This was confirmed both in vitro and ex vivo using parasites developing in the tsetse fly. Comparison of the results with the literature reveals that IFT transport adopts different configuration depending on the anatomy of cilia and flagella. This raises the question of the evolution of the transport system in association with that of organelle structure. Two intriguing possibilities will be discussed.
Adeline MALLET (Institut Pasteur)
16:00 - 18:00 #26230 - Field Evaporation Energy Loss Spectroscopy.
Field Evaporation Energy Loss Spectroscopy.

Loïc ROUSSEAU *, Felipe F. MORGADOLeigh STEPHENSONConstantinos HATZOGLOUKambiz TEHRANIAntoine NORMANDBaptiste GAULTFrançois VURPILLOT

  • Groupe Physique des Matériaux, Université de Rouen, Normandie, France
  • Max-Planck Institut für Eisenforschung GmbH, D-40237 Düsseldorf, Germany
  • Norges teknisk-naturvitenskapelige universitet (NTNU), Trondheim, Noway
  • ESIGELEC
  • Groupe Physique des Matériaux, Université de Rouen, Normandie, France


The design of new materials requires an in-depth exploration of their physical, or structural properties. Deciphering their structure and composition at the atomic scale is a real challenge. Atom Probe Tomography (APT) allows to map 3D composition of a specimen near the atomic scale for a wide range of materials. However, in APT, the information is restricted to the list of coordinates of atoms in the probed volume, with elemental information get from time-of-flight mass spectrometry. No real local chemical information is extracted from the data. To access it, APT data have been recently indirectly mined looking at the mechanism of field evaporation of atoms in thermoelectric and phase change materials[1]. Field evaporation is the fundamental mechanism involved to peel off the sample atom by atom. Deep inspection of the dynamic of field evaporation showed some differences from one sample to another related to the nature of the atomic bonding, which could help to explain their properties[2]. However, this information remained global and very indirect. In this paper, we show how energetic considerations in the field evaporation process are in fact intrinsically present within the data, but have until now been ignored or even consciously suppressed using ion optics devices. This information is indeed embedded inside mass spectra. Based on an analytical model and robust simulations, we confirm the validity of our method and document this parameter for a series of different materials. We finally demonstrate how this information can be mapped in 3D with nanometric resolution. A grain boundary example in a pure Al dataset and the case of recrystallisation of amorphous FeBSi metallic glasses will be presented (fig.1,2). We term this new approach ""Field Evaporation Energy Loss Spectroscopy"". FEELS can be adapted to any straight flight path APT using voltage pulses, and can even be used on the thousands of existing data sets to retrieve further information. [1] T. Ohnuma, Microsc. Microanal., pp. 1–7, 2021, doi: 10.1017/S1431927621000155. [2] M. Raghuwanshi et al., Nano Lett., vol.20, no.1, pp.116–121, 2020, doi: 10.1021/acs.nanolett.9b03435.
Loïc ROUSSEAU (Saint Etienne du Rouvray)
16:00 - 18:00 #27024 - THERMAL PROBE MICROSCOPY: TOWARD A THERMAL ANALYSIS OF NANOMATERIALS.
THERMAL PROBE MICROSCOPY: TOWARD A THERMAL ANALYSIS OF NANOMATERIALS.

Nathaly Chaaraou*, Patricia Al Alam, Nathalie Trannoy*, Jaona Randrianalisoa

University of Reims Champagne Ardenne, IThMM, Reims, 51687, France 



Nanotechnology requires characterization technics for nanosystems and nanomaterials. Since twenty years, Nanotechnology reveals new needs in understanding and management of heat distribution at micro/nanoscale and in nanoscale structures. Development in new materials requires advanced knowledge of nanoscale heat transfer and thermal properties of nanostructured materials. Scanning thermal microscopy (SThM) based on Atomic Force Microscopy (AFM) technique is a promising tool for investigating material’s thermal measurements and heat transfer mechanisms at the micro/nanoscale [1, 2]. This project aims to investigate temperature and/or thermal properties of the studied material using a SThM probe with thermal sensor (thermoresistive, thermocouple etc.) at the tip. This thermal tip allows us obtaining topographic and thermal images simultaneously with micrometer and sub-micrometer spatial resolution. In this matter, Wollaston is used as a resistive probe in order to: (i) study the influence of sample structure on the thermal signal of the probe, (ii) characterize and estimate the effect of probe volume on the thermal conductivity measurements. For that, samples composed of buried silicon steps under polished CVD SiO2. A well-defined heat transfer numerical model was developed for the probe-sample system with the surrounding medium using COMSOL Multiphysics based on finite element method. This simulation allows comparison between measured results and the model developed with all physical aspects considered. A 3D realistic geometry of the Wollaston probe is modelled in contact operation mode in order to obtain probe temperature behavior (Fig.1(a,b)). The probe/sample numerical model reveals the evaluation of the dissipated flux by the thermal resistive wire (platinum-rhodium wire) and into the sample. By comparing the experimental measurements with the simulation, it is clearly shown that the developed models are in good agreement with experiment results (Fig.2(a,b)) . In other words, the developed models are capable to rebuild experimental thermal profiles. More worthwhile , this study proofs the sensitivity of the thermal signal to the internal nanostructure of the investigated samples. Références/References: [1] Majumdar, Lai, Chandrachood, Nakabeppu, Wu and Shi, Thermal imaging by atomic force microscopy using thermocouple cantilever probes, 1995 [2] F.A. Guo, N. Trannoy, J. Lu, Analysis of thermal properties by scanning thermal microscopy in nanocrystallized iron surface induced by ultrasonic shot peening, Materials Science and Engineering: A, 2004.
Nathaly CHAARAOUI (Reims)
16:00 - 18:00 #26241 - Using Convolutional Neural Network for segmenting the nano-XCT Images of battery materials.
Using Convolutional Neural Network for segmenting the nano-XCT Images of battery materials.

Zeliang Su *, Etienne DescencièreTuan-Tu NguyenVincent De AndradeAlejandro A. FrancoArnaud Demortière

  • Laboratoire de Réactivité et Chimie des Solides (LRCS), CNRS UMR 7314, Université de Picardie Jules Verne, Hub de l’Energie, Rue Baudelocque, 80039 Amiens Cedex, France.
  • MINES ParisTech – PSL Research University, CMM, Center for Mathematical Morphology, 35 rue Saint Honoré, Fontainebleau, France
  • Argonne National Lab, Advanced Photon Sources Beam line, Bldg. 401/Rm A4115, Argonne National Laboratory 9700 S. Cass Ave. Argonne, IL 60439, USA.


X-ray tomography has been widely used in battery research in recent years for understanding the impact of the microstructure and the dynamics of the batteries. However, every experiment generates up to dozens of gigabytes, the quantity of image data is challenging for the segmentation. Additionally, for the nano-XCT data, where a high signal-to-noise ratio is challenging to obtain and a wide variety of artifacts is present, the traditional grayscale thresholding approach is not efficient enough for complex composite battery materials. Up to date, the task of segmentation is still tedious work. We, therefore, investigated in the Convolutional Neural Network (CNN Figure 1). We decrypted the working mechanism of an in-house CNN by visualizing the outputs of each unit and its gradients during the training. We reexamined the influence of each hyperparameter to determine the parameter tuning priority. The segmentation efficiency has been successfully demonstrated by giving only one example to train the CNN. And high accuracies have been obtained for various cathode material datasets for Li-ion and Li-O2 batteries[1][2]. To further reduce the computation time, we have also shown that transfer learning (using a well-trained CNN on a new dataset) could improve the convergence. A roof of accuracy has been observed during our experiments. Such a limit likely depends on the quality of the tomography and the experimenter. To figure out the impact of segmentation on the material properties determination, we used a simulation and statistical approach. Lastly, we have embedded our findings and CNN methods in open-source graphical software (Figure 2) to allow a broader community can benefit from training CNNs without knowing coding basics. Références/References : [1] Nguyen et al. Adv.Energy Mat. (2021) p. 2003529 [2] Su et al. ACS Appl. Energy Mater. 3 (2020), p.4093−4102 Figure 1: Raw nano-XCT volume of an NMC cathode material and the rendering of the segmentation results by CNN. At the bottom, slices of raw XCT-image a ground truth, and a prediction are showcased. Figure 2: The user-friendly interface of the open-source software SegmentPy for XCT segmentation
Zeliang SU (Amiens)
16:00 - 18:00 #26371 - IN SITU ANALYSIS OF THE PROTEIN CORONA BY CRYO-TRANSMISSION ELECTRON MICROSCOPY.
IN SITU ANALYSIS OF THE PROTEIN CORONA BY CRYO-TRANSMISSION ELECTRON MICROSCOPY.

Jéril DEGROUARD *, Laurent MARICHALStéphanie DEVINEAUGaël GIRAUDON-COLASYves BOULARDJean-Philippe RENAULTSerge PIN

  • Université Paris-Saclay, CNRS, UMR 8502, Laboratoire de Physique des Solides, 91405 Orsay Cedex, France
  • Université de Paris, BFA, UMR 8251, CNRS, F-75013 Paris, France
  • Université Paris-Saclay, CEA, CNRS, NIMBE, Gif-sur-Yvette 91190, France


In a biological environment, the adsorption of proteins on nanoparticles (NPs) is an early process that leads to major changes of the physicochemical properties and the biological effects of NPs. The formation of the so-called protein corona drives NP-cell interaction, NP biodistribution, and NP toxicity in vivo. However, few experimental techniques allow for the analysis of the protein corona in situ. As a result, little is known on its structure. Using silica NPs and a library of purified hemoproteins with sizes ranging from 2.8 to 22 nm in diameter (17 to 3,600 kDa in MW), we investigated the effect of protein size on the protein corona assembly by cryo-TEM. Our observations revealed that larger proteins, composed of more than a hundred subunits, form open networks with NPs rather than a homogeneous monolayer of proteins decorating the surface as observed for smaller ones (Figure 1) [1]. The ability of each protein to bind to several NPs drives the formation of novel supramolecular assemblies, while preserving protein structure and function, here in terms of oxygenation properties. By combining cryo-TEM and synchrotron radiation circular dichroism, we further showed that NPs can stabilize partially unfolded protein conformations, thereby driving the formation of a ‘soft protein corona’ consisting of small weakly bound proteins directly adsorbed to the surface, a dynamic process associated with the partial loss of protein structure and stability (Figure 2) [2]. These results highlight the need for in situ analytical approaches to investigate the structuring and driving forces of protein adsorption on nanomaterials to better our understanding of their effects in biologically relevant conditions. Acknowledgements: Cryo-TEM experiments were supported by the METSA network (Proposal FR3507) and by the French Investissements d’Avenir LabEx PALM (ANR-10LABX-0039- PALM). References : [1] From Protein Corona to Colloidal Self-Assembly: The Importance of Protein Size in Protein−Nanoparticle Interactions. Marichal L. et al. Langmuir (2020) 36, 8218-8230. [2] In situ analysis of weakly bound proteins reveals molecular basis of soft corona formation. Sanchez-Guzman D. et al. ACS Nano (2020) 14, 9073-9088.
Jéril DEGROUARD (Orsay)
16:00 - 18:00 #26374 - Structure determination of an artificial virus made of self-assembling cyclodextrin and dsDNA by cryo-EM.
Structure determination of an artificial virus made of self-assembling cyclodextrin and dsDNA by cryo-EM.

Mauricio G.S. Costa *, Zakaria L DahmaniCharline FagnenCarlos A.H. FernandesLeonid LavnevichCatherine Vénien-BryanMatthieu Sollogoub

  • Sorbonne Université, Muséum National d'Histoire Naturelle, Institut de Minéralogie, Physique des Matériaux et Cosmochimie, IMPMC, UMR 7590, CNRS, Paris, France
  • Sorbonne Université, Faculté des Sciences et d’Ingénierie, Institut Parisien de Chimie Moléculaire, Paris, France


Therapeutics based on siRNA are very promising. siRNA are very short double-stranded RNA with the ability to specifically inhibit the expression of a gene. This discovery is promising but meets some difficulties to be applied. One of the major obstacles is relative to their use as drugs, they need an efficient and non-toxic vector to let the siRNA reach its target and plays its role. A key challenge for their development is the design of sophisticated, safe and effective delivery methods. However, the development of such vectors for therapeutics on an industrial scale remains complicated and very expansive. Thus the imitation of their self-assembled highly cooperative structure with artificial systems is a real challenge. Taking into consideration this fact, we synthetized an artificial virus inspired by the tobacco mosaic virus to bring the siRNA to the targeted cell. Complexes cyclodextrin/siRNA (CD-siRNA) and CD-dsDNA were realized in fiber form. Our goal is to understand the assembly mechanism between the cyclodextrin and the cdDNA, for that an electron microscopy study has been led on this complex to resolve this structure at high-resolution. The complexation of CD-dsDNA was checked by electrophoresis studies. The fiber synthetized is a polymer of very big molecur weight. In order to determine the 3D structure of the complex, it is necessary to choose a method able to deal with big sizes systems. It is for this reason that cryo-EM was chosen to build the structure in 3D to understand the assembly mechanism of the complex CD-dsDNA. The 3D structure of these fibers made of CD-dsDNA at high resolution was calculated from cryo-EM images combined witrh image analysis. This structure gives some clues on the organization of the cyclodextrins and their interaction with the siRNA. Reference: 1. Kanasty, R., et al.. Delivery materials for siRNA therapeutics. Nat Mater 12, 967–977 (2013)
Mauricio G.s. COSTA (Sorbonne Université)
16:00 - 18:00 #26375 - Combined Hamiltonian Monte Carlo and Normal Mode Flexible Fitting.
Combined Hamiltonian Monte Carlo and Normal Mode Flexible Fitting.

Rémi Vuillemot *, Slavica JONIC

  • IMPMC - UMR 7590 CNRS, Sorbonne Université, MNHN, Paris, France


Density volumes obtained by three-dimensional reconstruction of biomolecular complexes from cryogenic electron microscopy (cryo-EM) images (also known as cryo-EM maps) can be interpreted in terms of atomic positions by flexible fitting. The fitting modifies an available atomic structure to match the target EM map. The most accurate fitting methods are based on atomic-coordinate degrees of freedom (e.g. Molecular Dynamics (MD), Monte-Carlo Simulation) but come with high computational cost for large required displacements. To reduce the computational cost, methods based on Normal Modes Analysis (NMA) decrease the number of degrees of freedom to only several collective motions (described by normal modes). The NMA-based methods are well-suited for global atomic displacements (large collective motions) but are suboptimal regarding local atomic displacements. To take advantages of both methods, we propose to combine them. More precisely, we combine Hamiltonian Monte Carlo and normal mode flexible fitting. We tested our method using synthetic and experimental cryo-EM maps of several complexes with large-scale conformational changes. Fig.1 shows the result of the fitting of a synthetic cryo-EM map of p97 ATPase. It shows that the combination of both approaches efficiently performs global and local atomic displacements and that it is more efficient and precise than any of the two approaches alone. The presentation will include experimental cryo-EM map fitting examples. To the best of our knowledge, this is the first flexible fitting method combining a Bayesian approach based on Hamiltonian Monte Carlo sampling and normal mode analysis.
Rémi VUILLEMOT (Paris)
16:00 - 18:00 #26376 - Tundra Cryo-TEM: Cryo-TEM that is cost effective and easy to use, bringing cryo-electron microscopy to every biochemistry laboratory.
Tundra Cryo-TEM: Cryo-TEM that is cost effective and easy to use, bringing cryo-electron microscopy to every biochemistry laboratory.

Massimiliano Maletta *

  • TFS


One of the primary challenges of investigating dynamic biological processes, and how they fail in disease, is the complexity inherent to biological machinery. Large and/or dynamic protein systems present a unique challenge to traditional methods of scientific inquiry, which study these systems either indirectly or in isolation. Fortunately, cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has emerged as a well-suited approach for the direct determination of native function and dynamics in complex biological systems. Single particle analysis can validate your biochemistry work by showing the molecular details that underlie the interactions between proteins, small molecules, and post-translational modifications in large and dynamic protein systems at near native conditions. These molecular details confirm the mechanism of action by which complex biological systems (e.g. membrane proteins, protein complexes, and macromolecular machines such as viruses, ribosomes, and proteasomes) contribute to human health and disease. For example, the human GABAA (gamma-aminobutyric acid type A) receptor is a small membrane protein and ligand-gated chloride-ion channel that mediates inhibitory neurotransmission. GABAA receptors are important therapeutic targets as their various conformations affect a variety of important signaling pathways. Due to GABAA's conformational flexibility, traditional methods have been unable to reveal its molecular mechanism of action. The ability of cryo-EM to image large, biologically complex structures has, meanwhile, allowed researchers to see the molecular details that underpin the allosteric modulation of this important receptor. With the Tundra Cryo-TEM, more scientists can have access to this type of information for their complex biological systems. Our applications scientists have used the Tundra Cryo-TEM to determine a 3D structure of GABAA to ~4-5 Å resolution. At this resolution, molecular details such as individual alpha helices and beta sheets are clearly visible, molecular structures can be docked, and molecular interactions modeled. Additionally, the Tundra Cryo-TEM was able to achieve 3 Å resolution for the rigid benchmark protein apoferritin. At this resolution, the protein backbone can be traced, and major amino acid side chains are clearly visible, making de novo model building possible. Data at these resolutions helps you understand how proteins function, how to modify genes, and how to design drugs accordingly.
Max MALETTA (, )
16:00 - 18:00 #26377 - Cryo-EM structure of the yeast SWI/SNF complex.
Cryo-EM structure of the yeast SWI/SNF complex.

Maximilien Werderer *, Adam Ben-ShemGabor PapaiCorinne CrucifixPatrick Schultz

  • Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France


Diploid human cells contain 2 meters of DNA condensed within a 5 μm nucleus. This condensed DNA is not accessible to the transcription machinery, and must be spatially organised to allow the decompaction and expression of specific genes when deemed necessary by the cell. DNA is packaged into chromatin and its level of compaction is regulated by protein complexes called chromatin remodelers. One of these chromatin remodeling complexes is the switching defective/sucrose nonfermenting (SWI/SNF) complex. It is highly conserved from yeast to human and regulates many critical cellular processes, including gene expression, cellular differentiation and DNA repair by using the energy of ATP to remodel the structure of the chromatin. SWI/SNF has recently been identified as a tumor suppressor in human malignancies as proteomics studies revealed that the complex subunits of the complex are mutated in almost 20% of all human tumors leading to aberrant SWI/SNF complexes [1]. However, the mechanical details of the SWI/SNF remodelling activity remain poorly understood, hindered by the lack of high resolution structure of the complex. Cryo-Electron Microscopy (Cryo-EM) is used to investigate the structure of proteins and complexes by imaging hydrated samples in vitrous ice in a transmission electron microscope. Recent advances in hardware and software allows Cryo-EM to image multi-protein complexes at sub-nanometer resolution and solve their atomic structure. Here we present the structure of the yeast SWI/SNF complex from the yeast Pichia pastoris obtained through Cryo-EM at near atomic resolution (Fig. 1). The high degree of structural conservation between yeast and human allow us to map mutations identified in human cancers and provides new insight into mechanistic implication of these mutations (Fig. 2). References: [1] Kadoch, C., Hargreaves, D. C., Hodges, C., Elias, L., Ho, L., Ranish, J., & Crabtree, G. R. (2013). Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nature Genetics, 45(6), 592–601. https://doi.org/10.1038/ng.2628
Maximilien WERDERER (Illkirch)
16:00 - 18:00 #26378 - Structural insights into how bS1 promotes translation.
Structural insights into how bS1 promotes translation.

Gaetano D'Urso *, Sophie ChatReynald GilletEmmanuel Giudice

  • Université Rennes, CNRS, Institut de Génétique et Développement de Rennes (IGDR) UMR 6290, Rennes, France


In prokaryotes, the initiation of the translation is known to be directed by the mRNA Shine – Dalgarno (SD) sequence [1]. However, some mRNAs contain a weak or even no SD [2]. In these cases, the bS1 protein has been show to efficiently promote the initiation of translation [3, 4]. bS1 is a bacterial ribosomal protein composed of six domains (D1 to D6). The N-terminal end (D1 – D2) interacts with the 70S [4-6] while the rest of the protein (D3 to D6) forms a flexible arm catching mRNAs [7]. In the present study we characterized, using cryo-EM, the role of E. coli bS1 protein during the initial steps of translation (Figure 1). In agreement with the previous founding, the interaction is primarily based on the stacking between bS1 and 70S uS2 (Figure 1). Surprisingly, the D2 assumes a new conformation thanks to a partial misfolding of the helix connecting the two domain, placing an hydrophobic pocket just upstream the SD – anti SD base pairing. These results suggest the involvement of D2 in the correct positioning of mRNA into the ribosome and in the stabilization of the SD – anti SD base pairing, as well as its fundamental role in contacting the ribosome. References: [1] Shine J, Dalgarno L. Proc Natl Acad Sci USA. 71 (1974), p. 1342-6 [2] Marzi, S. et al. Myasnikov, A. G., Serganov, A., Ehresmann, C., Romby, P., Yusupov, M., Klaholz, B. P. Cell Press. 130 (2007), p. 1019-1031 [3] Irina V. Boni, Dilara M. lsaeva, Maxim L. Musychenko, Nina V. Tzareva, Nucleic Acids Research, 19 (1991), p. 155–162 [4] Salah P, Bisaglia M, Aliprandi P, Uzan M, Sizun C, Bontems F. Nucleic Acids Res. 37 (2009), p. 5578-88. [5] Loveland, A. B., Korostelev, A. A. Methods. 137 (2018), p. 55-66 [6] Byrgazov K, Grishkovskaya I, Arenz S, Coudevylle N, Temmel H, Wilson DN, Djinovic-Carugo K, Moll I. Nucleic Acids Res. 43 (2015), p. 661-73 [7] Duval, M., Korepanov, A., Fuchsbauer, O., Fechter, P., Haller, A., Fabbretti, A., Choulier, L., Micura, R., Klaholz, B. P., Romby, P., Springer, M., Marzi, S. PLOS Biology. 11 (2013), e1001731
Gaetano D'URSO (Rennes)
16:00 - 18:00 #26381 - Mode de processivité de la formine mDia1 aux extrémités barbées des filaments d'actine élucidé par microscopie électronique.
Mode de processivité de la formine mDia1 aux extrémités barbées des filaments d'actine élucidé par microscopie électronique.

Julien Maufront *, LuYan CaoBérengère GuichardDaniel LevyAntoine JégouGuillaume Romet-LemonneAurélie Bertin

  • Institut Curie UMR168
  • Institut Jacques Monod


Les Formines stimulent la nucléation et l'élongation rapide des filaments d'actine non branchés dans les cellules. À l’échelle moléculaire, elles constituent des homodimères capables d’interagir avec les extrémités barbées des filaments d’actine en élongation via leurs domaines FH2. Bien que les changements conformationnels impliqués dans ce processus n'aient jamais été observés directement, des études suggèrent que le dimère de FH2 se déplace pas à pas à l'extrémité barbée à travers un équilibre entre deux états dits « fermé » et « ouvert ». Plusieurs modèles ont pu être proposés pour décrire ces deux états conformationnels ainsi que la transition de l’un vers l’autre comme le modèle de la « marche enjambée » ou le modèle du « saut enclenché ». Aucune structure en microscopie électronique n’avait été obtenue jusqu’à présent pour lever ces interrogations. En effet, rapidement, en présence de formines, de longs filaments d’actine sont générés et ne permettent pas l’obtention d’une densité élevée de courts filaments coiffés de formines, nécessaire à une analyse en particules isolées. Un goulot d'étranglement a donc dû être surmonté afin de rendre possible une telle analyse avec notamment le choix d’une approche par sonication. Deux structures 3D du complexe bout barbé - formine dans sa conformation majoritaire et minoritaire ont pu être déterminées en coloration négative. Ces deux conformations correspondent respectivement aux états « ouvert » et « fermé » décrits par le modèle de la « marche enjambée » et confortent ainsi ce dernier modèle. Des variations conformationnelles au sein de l’état « ouvert » ont également pu être mise en évidence au cours d’une analyse 2D focalisée. Ces observations ont permis de proposer l’existence d’un chemin d’interaction à l’issu duquel le dimère de FH2 encercle le corps du filament d’actine, en amont de l’extrémité. Cette hypothèse a pu être validée par une analyse 2D suivie d’une reconstruction 3D. La transposition de ces résultats à une approche par cryo-microscopie a été entreprise via l’utilisation de grilles recouvertes d’oxide de graphène. Une quantité limitée d’extrémités a pu être analysée en 2D et 3D confirmant à minima la nature non artéfactuelle des résultats précédents.
Julien MAUFRONT (Paris)
16:00 - 18:00 #27356 - Structural study of the tail tip of siphophage T5.
Structural study of the tail tip of siphophage T5.

Romain LINARES, Grégory EFFANTIN, Charles-Adrien ARNAUD, Claudine DARNAULT , Elisabetta BOERI ERBA , Guy SCHOEHN, Cécile BREYTON

Institut de Biologie Structurale, 71 avenue des Martyrs, CS 10090, 38044 Grenoble Cedex 9, FRANCE



The vast majority (96%) of bacteriophages - bacterial viruses - possess a tail that allows host recognition, cell wall perforation and safe viral DNA channelling from the capsid to the cytoplasm of the infected bacterium. The majority of tailed phages, the Siphoviridae, bear a long flexible tail formed of stacked tail tube proteins (TTP) that polymerise around and along the tape measure protein (TMP). At the distal end of the tail, the tail tip complex harbours the receptor binding protein·s (RBP). Interaction between the RBP with the host surface triggers cell wall perforation and DNA ejection, but little is known on these mechanisms for Siphoviridae. We aim at characterising the mechanism of cell wall perforation and DNA ejection in the siphocoliphage T5. We have determined the structure of T5 TTP and tail tube, before and after interaction with its E. coli receptor, and shown that host binding information is not propagated to the capsid by the tail tube [1]. We now focus on the structure of the tail tip complex, and we will present high-resolution structures of T5 tail tip complex, determined by cryo-electron microscopy and single particle reconstruction on purified T5 tails. We could trace 6 of the 7 proteins that compose it [2, 3], and we will discuss the structural homology with other phages and phage-derived complexes Références/References : [1] Arnaud C-A, Effantin G, Vivès C, Engilberge S, Bacia M, Boulanger P, Girard E, Schoehn G and Breyton C, Bacteriophage T5 tail tube structure suggests a trigger mechanism for Siphoviridae DNA ejection Nat Com, 8, 1953 (2017) [2] Zivanovic, Y, Confalonieri, F, Ponchon, L, Lurz, R, Chami, M, Flayhan, A, Renouard, M, Huet, A, Decottignies, P, Davidson, AR, Breyton, C, and Boulanger, P, Insights into bacteriophage T5 structure from the analysis of its morphogenesis genes and protein components. J. Virol. 88:1162-74 (2014) [3] Flayhan, A, Vellieux, FMD, Lurz, R, Maury, O, Contreras-Martel, C, Girard, E, Boulanger, P, and Breyton, C Crystal structure of pb9, the distal tail protein of bacteriophage T5: A conserved structural motif among all siphophages. J. Virol. 88, 820-8 (2014)
Romain LINARES (Grenoble)
16:00 - 18:00 #26356 - ORGANISATION SUPRAMOLECULAIRE DANS LES FIBRES DE COLLAGÈNE.
ORGANISATION SUPRAMOLECULAIRE DANS LES FIBRES DE COLLAGÈNE.

Amélie LEFORESTIER *, Gervaise MOSSERJéril DEGROUARDJean CHARVOLINJean-François SADOC

  • Université Paris-Saclay, CNRS UMR8502, Laboratoire de Physique des Solides, 91405, Orsay, France
  • Sorbonne Université, CNRS UMR 7574, Laboratoire de chimie de la matière condensée de Paris, 75005 Paris


Le collagène est le principal polymère de la matrice extracellulaire chez les vertébrés. Il joue un rôle essentiel dans l’intégrité des tissus, dans leurs propriétés mécaniques (peau, tendon, cartilage) ou optiques (cornée). La matrice de collagène présente une structure hiérarchique : les triples hélices forment des fibrilles striées de diamètre variable (20-200 nm), qui elles-mêmes présentent différents types d’arrangements supra-fibrillaires. Malgré de nombreuses études, la structure des fibrilles est très mal connue. Le modèle d’organisation longitudinale échelonnée [1], explique de façon simple les striations du collagène de type I, mais ne contribue pas à une description de l’organisation 3D. Différents modèles théoriques ont été proposés, sans que la diffusion des rayons X ne permette de trancher, du fait d’une coexistence structurale d’ordre et de désordre : i) Une organisation poly-cristalline de micro-fibrilles pentagonales aplaties avec des lignes de dislocations radiales [2]. ii) Une organisation phyllotactique [3] (Figure 1A) qui présente une coexistence ordre-désordre intrinsèque et assure une densité optimale par ses lignes de dislocations circulaires (cercles verts et bleues). En nous appuyant sur un ensemble de techniques de microscopie, nous analysons l’organisation du collagène au sein des tendons de souris. En particulier, nous analysons l’organisation latérale des triples hélices au sein des fibrilles en utilisant la cryo microscopie électronique de sections vitreuses (CEMOVIS). Nous montrons que les triples hélices de collagène peuvent être ainsi visualisées (Figure 1C, D). Un ordre hexagonal local est mis en évidence (Figure 1C, C’). Nous analysons le facettage des fibrilles, l’ordre local et la torsion entre molécules. A plus long terme, les impacts de différentes pathologies sur ces organisations pourraient être mise en évidence et révéler ainsi des déstructurations aux échelles nanométriques. Références : [1] Petruska J.A., Hodge A.J. (1964) Proc. Natl. Acad. Sci. USA 51, 871. [2] Hulmes D.J. (2002). J. Struct. Biol., 137, 2-10. [3] Charvolin J., Sadoc J.F. (2014) Biophysical Reviews and Letters, 9, 225-238 ; Charvolin J., & Sadoc J.F. (2013) Biophysical Reviews and Letters, 8, 33-49.
Amélie LEFORESTIER (Paris-Saclay)
16:00 - 18:00 #26358 - Order and disorder in DNA toroids analyzed by cryo electron tomography.
Order and disorder in DNA toroids analyzed by cryo electron tomography.

Kahina VERTCHIK *, Cédric MESSAOUDISylvain TREPOUTAmélie LEFORESTIER *

  • Université Paris-Saclay, CNRS, Laboratoire de Physique des Solides, 91405 Orsay, France.
  • Multimodal Imaging Center, Centre Institut Curie, PSL Research University, CNRS, Université Paris-Saclay, INSERM, 91401 Orsay, France.


DNA toroids are complex nanometer-scale liquid crystalline objects that combine properties of both biological and material sciences objects. They spontaneously form in vitro in the presence of spermine 4+ that condenses DNA in solution. In these condensates, DNA strands locally organize with hexagonal packing, but topological constraints impose the existence of defects. Deviation from hexagonal order arise from the continuity of the DNA chain that creates crossover defect [1] and from the competition between twist induced by the DNA helices and the parallelism of hexagonal lattice which creates frustration [2]. In order to investigate the links between the macroscopic and microscopic structure of toroidal condensates, DNA toroids obtained from bacteriophage ejecting their genome in the presence of spermine [3] were investigated using cryo electron tomography. Tomogram reconstruction allowed a direct visualization of the hexagonal lattice of the DNA helices. However, Bragg contrast precludes unambiguous analyses in most regions. We therefore analyze local order in 2D images extracted from the tilt series in relationship with the overall shape of the condensate obtained in the reconstructed tomogram. Segmentation of the toroids reveals the existence of a disordered region, less dense that hexagonally packed regions. These regions could be related to bridge defects predicted by simulations [4] and/or concentration of DNA ends. A faceting of toroids is also highlighted. We analyze the relationship between the rotation of the lattice [2] and the faceting of the toroid. References: [1] Hud N.V., Downing K.H. (2001) Proc. Natl. Acad. Sci. USA 98, 14925-14930. [2] Leforestier A., Livolant F. (2009) Proc. Natl. Acad. Sci. USA, 106, 9157-9162. [3] Barberi L., Livolant F., Leforestier A., Lenz M. (2020) Nucleic Acid Res., in press. [4] Dey A., Reddy G. (2017) J. Phys. Chem B, 121, 9291.
Kahina VERTCHIK (Orsay)
16:00 - 18:00 #26360 - Deciphering chromatin organization in situ using cryo-electron tomography and image denoising techniques.
Deciphering chromatin organization in situ using cryo-electron tomography and image denoising techniques.

Fadwa FATMAOUI *, Diana GREWEAmelie LEFORESTIERMikhail ELTSOV

  • Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch
  • Buchmann Institute for Molecular Life Sciences, Frankfurt
  • Laboratoire de Physique des Solides, Orsay


Cryo-electron tomography (cryo-ET) has recently emerged as one of the main methods for high-resolution characterization of biological structures. While single-particle approaches require averaging images of a number of identical structures, cryo-ET allows direct three-dimensional imaging of biological structures with nanometer resolution in situ, which is particularly advantageous for studying complex pleomorphic structures. We apply in situ cryo-ET to analyze chromatin, which exhibits a high degree of heterogeneity at all levels of its organization, starting with nucleosome particles [1]. Tomographic resolution is directly dependent on sample thickness, so we use vitreous cryosectioning (CEMOVIS, [2]) to obtain < 100 nm thick slices from vitrified Drosophila embryos to provide visualization of fine structural details (Figure 1).The second major obstacle to the structural interpretation of tomographic reconstructions is the very low signal-to-noise ratio. The conventional solution to this problem is based on averaging of subvolumes to remove noise, but it cannot be applied to pleomorphic chromatin fibers. We demonstrate that recent denoising algorithms based on deep learning [3] can effectively deal with noise in our cryo-ET data, thereby providing visibility of fine structural details of in situ chromatin organization (Figure 2). Acknowledgements: We acknowledge the support of the French National Research Agency (ANR-20-CE11-0020-02 to ME, ANR-20-CE11-0020-01 to AL); German Research Foundation (DFG EL 861/1 to ME). References: [1] Eltsov et al., Nucleic Acids Research, 46 (2018), p. 9189–9200 [2] Al‐Amoudi et al., EMBO Journal, 23(2004), p. 3583-3588 [3] Tegunov and Cramer, Nature Methods, 16(2019), p. 1146–1152
Fadwa FATMAOUI (Illkirch)
16:00 - 18:00 #26361 - Nanometric Probes for Cryo-Tomography Analysis.
Nanometric Probes for Cryo-Tomography Analysis.

Victor Hanss *, Guy ZuberMikhail EltsovPatrick Schultz

  • Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Integrated Structural Biology, Centre National de la Recherche Scientifique (CNRS), UMR 7104, Institut National de la Santé et de la Recherche Médicale (INSERM), U1258
  • Ecole Supérieure de Biotechnologie Strasbourg (ESBS), Biotechnologie et Signalisation Cellulaire, Centre National de la Recherche Scientifique (CNRS), UMR 7242, Université de Strasbourg, lllkirch, France.


In vitro macromolecular structure analysis methods do not represent the macromolecules architecture under cellular constrains. We therefore aim at observing biomolecules within vitrified cellular sections by cryo electron tomography. However, transient molecular partners, which can be small and rare, are difficult to identify. To overcome this barrier, we want to label macromolecules of interest within human cells with antibodies conjugated to gold nanoparticles (AuNp) before cell vitrification. Structure studies on in vivo labelled molecules requires the following steps (Fig. 1). First, electron dense AuNps are conjugated to a probe and electroporated into cells [1]. Cryo-fixation is then performed by plunge freezing or high pressure freezing. Since only less than 300 nm thick samples can be used for sub-molecular structure analysis, the sample needs to be thinned [2]. : Cryo-EM of vitreous sections (CEMOVIS) generates thin sections of the cryo-fixed sample [3]. Alternatively focused ion beam (FIB) thinning mills thin lamellae of the vitrified samples directly on the EM grid [2]. Here we show preliminary data of antigen binding fragment (Fab) conjugated with 2,4 nm diameter AuNps directed towards the Human RNA polymerase DNA dependant II C-terminal domain (RNAPII-CTD). In order to control the fab conjugate efficiency and specificity we fixed, permeabilized and incubated Hela cells with anti RNAPII-CTD mice Fabs (fab@RNAPII-CTD). A fluorescent detection in immunohistochemistry was performed with a second antibody coupled to the Alexa488 fluorophore (@mouse-Alexa488). The Alexa488 signal from the fab incubated Hela cells overlaps with Hoechst’s nucleus stain (Fig. 2, 1 b,e) while the weak signal from buffer incubated cells is not (Fig. 2, 1 d,f). The same was observed after silver enhancement of AuNps within incubated Hela cells (Fig. 2, 2). Fab@RNAPII-CTD incubated cells show a strong nuclear signal after silver enhancement. These preliminary data suggest that the gold-conjugated fab kept its specificity towards RNAPII-CTD after AuNp conjugation. References: [1] N. Groysbeck et al., Nanotechnology, vol. 30 (2019) p. 184005 [2] M. Schaffer, et al., Journal of Structural Biology, vol. 197 (2017) p. 73‑82 [3] A. Al-Amoudi et al., EMBO J, vol. 23 (2004) p. 3583‑3588
Victor HANSS (Illkirch)
Room 1