Optimizing cryogenic super-resolution microscopy for integrated structural biology
Super-resolution fluorescence microscopy (“nanoscopy”) enables biological imaging at the nanoscale. This technique has already revolutionized cell biology, and today it enters the field of structural biology. One major evolution concerns the development of nanoscopy at cryogenic temperature (“cryo-nanoscopy”). Cryo-nanoscopy offers several key advantages, notably the prospect of an extremely precise correlation with cryo-electron tomography (cryo-ET) data. However, cryo-nanoscopy has not provided super-resolved images of sufficiently high quality yet. This PhD project will focus on the optimization of cryo-nanoscopy using the Single Molecule Localization Microscopy (SMLM) method with fluorescent proteins (FPs) as markers. Our goal is to significantly improve the quality of achievable cryo-SMLM images by (i) engineering and better understanding the photophysical properties of various FPs at cryogenic temperature, (ii) modifying a cryo-SMLM microscope to collect better data and (iii) developing the nuclear pore complex (NPC) as a metrology tool to quantitatively evaluate cryo-SMLM performance. These developments will foster cryo- correlative (cryo-CLEM) studies linking cryo-nanoscopy and cryo-FIB-SEM-based electron tomography.
In vitro reconstitution of microtubule network polarization.
Microtubules, biological polymers present in all eukaryotic cells, serve as a support for intracellular transport via molecular motors, thus defining cellular polarity. Contrary to the dogma establishing the centrosome as the determinant of this polarity, research from the CytoMorpho Lab reveals that microtubules can self-organize without an organizing center. In vitro experiments have demonstrated that microtubules actively separate molecular motors of opposite polarities into distinct domains, creating a new mechanism of active phase separation. Such partitioning of space by microtubules and motors constitutes a new mechanism of morphogenesis. The doctoral project aims to encapsulate this system in lipid vesicles of controlled size to study how relative dimensions enable efficient polarization. This approach will require the development of a microfluidic device and optimization of biochemical conditions for anchoring motors in the lipid bilayer. The perspectives include the creation of "artificial cells" capable of polarization and the reevaluation of cellular polarization models, particularly for T lymphocytes and other differentiated cells.
Study of the thermomechanical properties of solid hydrogen flows
IRIG's Department of Low Temperature Systems (DSBT) is developing several research themes around cryogenic solid hydrogen and its isotopes. The applications of this research range from the production of renewable micrometre-sized solid hydrogen targets for the generation of high-energy protons for laser-plasma acceleration, to the formation and injection of millimetre- or centimetre-sized hydrogen ice cubes for the supply and control of plasma in fusion reactors using magnetic or inertial confinement. A cross-cutting issue in these applications is the need for a detailed understanding of the mechanical properties of solid hydrogen, in order to gain a better understanding of the physics of extrusion and target production, as well as the formation and acceleration of icicles for injection into fusion plasmas.
The subject of this thesis focuses on the study of solid hydrogen extrusion under pressure. Using this technology, the DSBT has been developing several cryostats for over 10 years, enabling the production of ribbons of solid hydrogen, ranging in size from a few millimetres to a few tens of micrometres, extruded at speeds of a few millimetres per second.
The main objective of the research is to gain a better understanding of extrusion mechanisms to enable the development of numerical predictive tools for extrusion system design. This experimental thesis will be based on cryogenic rheometry using a capillary rheometer and/or a duvet experiment developed during a previous thesis. This study will be carried out in collaboration with the Laboratoire de Rhéologie et Procédés at Grenoble Alpes University.
INVESTIGATION OF CONFORMATIONAL HETEROGENEITY AND DYNAMICS IN FLUORESCENCE ACTIVATING AND ABSORPTION-SHIFTING TAGS (FAST)
Fluorescent proteins, particularly Reversibly Switchable Fluorescent Proteins (RSFPs), have revolutionized advanced fluorescence imaging, paving the way for applications such as super-resolution microscopy. Among emerging alternatives, fluorogen-based reporters, such as the FAST (Fluorescence Activating and Absorption Shifting Tag) system, stand out dur to their enhanced photostability and versatility. FAST operates via non-covalent binding of a small engineered protein to an organic fluorogen, which induces fluorescence and allowing real-time monitoring without chromophore maturation. However, challenges remain in optimizing these systems due to limited mechanistic understanding of fluorogen-protein interactions, binding dynamics, and photophysical behavior under illumination. This PhD project aims to characterize the binding modes of FAST systems at atomic resolution using multidimensional NMR spectroscopy, X-ray crystallography, and UV-visible spectroscopy. Recent findings suggest that fluorogens can adopt multiple binding modes, and that slight chemical modifications impact binding kinetics and fluorescence brightness. By integrating laser-based illumination in NMR investigations, we will further probe how light absorption affects fluorogen conformation and dynamics. The insights gained from this study will enable the rational design of optimized FAST variants, enhancing their performance for specific microscopy applications and advancing the field of fluorescence imaging.
Molecular dynamics and disorder in the viral replication machinery of SARS CoV 2
The nucleocapsid protein (N) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for genome replication, encapsidating the viral genome and regulating gene transcription. The protein is highly disordered, comprising two disordered termini and a central disordered domain that are essential to its function. The central domain contains a number of important mutations that are responsible for enhanced viral fitness, and comprises a region that is hyperphosphorylated during the viral cycle. NMR spectroscopy is the tool of choice for studying the conformational behaviour of intrinsically disordered proteins, an abundant class of proteins that are functional in their disordered form. They represent 40% of the proteome and are too dynamic to be studied by crystallography or electron microscopy. The host lab has developed a large number of unique NMR-based tools to help understand the function of this class of proteins at atomic resolution. We will use NMR, paramagnetic NMR, small angle scattering, single molecule FRET and electron microscopy, in combination with molecular dynamics simulation, to describe the interactions of N with viral partner proteins and viral RNA to describe the process of encapsidation of the viral genome by the nucleocapsid protein, as well as the impact of mutations present in variants of concern. The results will be correlated with light and electron microscopy, carried out in collaboration.
Chiral Superconductors and Thermal Transport
In this PhD project, we intend to probe two well-known unconventional superconductors with thermal transport, through an original approach combining macroscopic and microscopic probes. These superconductors are UPt3 and UTe2, chosen because they address two issues currently under hot debate in the international community, that could strongly benefit from this new approach. UPt3 addresses the question of topological superconductivity, while UTe2 requires a clear identification of its spin-triplet superconducting order parameter.
Topological superconductivity is an active subject on the theoretical side and because of its potential interest in the field of quantum engineering. However, unambiguous experimental results are scarce, and we intend to focus here on UPt3, the first ever superconductor demonstrating the existence of transitions between superconducting phases, together with convincing evidences for chiral superconductivity. The goal is to probe predictions on the existence of an anomalous (zero field) thermal Hall effect, which would arise from the chiral edge currents.
A new approach is proposed, combining a newly designed set-up for the macroscopic measurement of thermal conductivity and thermal Hall effect, together with a microscopic probe realizing Scanning Thermal Spectroscopy. This will be realized thanks to a collaboration between two labratories in Grenoble: a team Pheliqs, mastering high quality crystal growth of these systems together with low temperature thermal transport measurements, and two teams in Néel, experts in Scanning SQUID microscopy and microscopic thermal measurements down to sub-Kelvin temperatures.
With this project, the PhD student will acquire very broad skills, ranging from sample preparation, low temperature instrumentation, and major actual issues in the field of quantum materials.
Control of two-dimensional magnetism by structural and chemical engineering of van der Waals interfaces
2D materials exhibit tunable interlayer interactions due to weak van der Waals bonding, which influences magnetic ordering in 2D magnets. The stacking sequence and internal chemistry impact ferromagnetic (FM) or antiferromagnetic (AFM) ordering, as seen in materials like CrBr3, CrI3, and Fe5GeTe2, where doping with Co raises the Curie temperature and alters magnetic phases. Chemical disorder also affects magnetic properties, with Mn/Sb substitution promoting FM ordering in Mn(Bi,Sb)2Te4. However, understanding how the atomic structure affects macroscopic magnetic properties remains limited due to the coexistence of metastable configurations. Precise control over stacking and chemical order is needed to harness 2D materials' magnetic and quantum properties. Transmission electron microscopy (TEM), especially aberration-corrected STEM, is today one of the most powerful techniques, enabling atomic-scale imaging and spectroscopy, for studying structural and chemical properties of 2D materials. This PhD project aims to study the relationship between atomic structure, chemistry, and magnetic properties in epitaxial 2D layers like (Fe,Co)5GeTe2, combining growth via molecular beam epitaxy (MBE) with STEM-based structural and chemical analysis.
Unraveling the mechanism of enzymatic carbon fixation
The Synchrotron Group at the Institut de Biologie Structurale in Grenoble is currently developing an innovative method called TR-FOX (Time-Resolved Functional Oscillation Crystallography). This technique aims to elucidate, firstly, the global dynamics of biological macromolecules in action and, secondly, their fine catalytic mechanism. It relies on the use of an injector capable of depositing onto the crystal, during the course of the X-ray diffraction data collection, a nanoliter droplet containing the substrate and cofactor of the studied reaction. This triggers the enzymatic reaction within the crystal. The approach can be combined with UV-Visible absorption spectroscopy to characterize the reaction kinetics more precisely. The goal is to obtain a series of structures during the catalytic cycle in order to make a molecular movie depicting the functioning of the enzyme. This thesis has two objectives: (i) improve and validate the TR-FOX method and, (ii) study the catalytic mechanism of two enzymes involved in carbon fixation either by capture or conversion of CO2.
Magnetic DIsks as Transducer of Angular Momentum
The proposed topic is a collaborative project to exploit suspended magnetic disks as novel microwave transducers of orbital angular momentum. Our goal is to develop ultra-high fidelity opto-mechanical modulators operating at GHz frequencies by integrating magnetic materials into optical components. This innovative concept arises from recent progress in the study of angular momentum conservation laws by magnon modes in axi-symmetric cavities, leading to new opportunities to develop a more frugal, agile, and sustainable communications technology. Our proposed design has the potential to achieve coherent interconversion between the microwave frequency range in which wireless networks or quantum computers operate and optical network frequencies, which is the optimal frequency range for long-distance communications. In this regard, our proposal not only proposes new applications of magnonics to the field of optics not previously envisioned, but also builds a bridge between the spintronics and the electronic and quantum communities.
In this proposal, the elastic deformations are generated by the magnetization dynamics through the magneto-elastic tensor and its contactless coupling to a microwave circuit. We have shown that coherent coupling between magnons and phonons can be achieved by precisely tuning the magnetic resonance degenerate with a selected elastic mode via the application of an external magnetic field. We expect to achieve ultra-high fidelity conversion by focusing our study on micron-sized single crystal magnetic garnet structures integrated with GaAs photonic waveguides or cavities. In addition, we propose the fabrication of suspended cavities as a means to minimize further energy leakage (elastic or optical) through the substrate.
The first challenge is to produce hybrid materials that integrate high quality garnet films with semiconductors. We propose a radically new approach based on micron-thick magnetic garnet films grown by liquid phase epitaxy (LPE) on a gadolinium-gallium-garnet (GGG) substrate. The originality is to bond the flipped film to a semiconductor wafer and then remove most of the the GGG substrate by mechanical polishing. The resulting multi-layer is then processed using standard lithography techniques, taking advantage of the relative robustness of garnet materials to chemical, thermal or milling processes.
The second challenge is to go beyond the excitation of uniform modes and target modes with orbital angular momentum as encoders of arbitrarily large quanta of nJ? for mode multiplexed communication channels or multi-level quantum state registers. The project will take advantage of recent advances in spin-orbit coupling between azimuthal spin waves as well as elastic scattering of magnons on anisotropic magneto-crystalline tensors. In this project, we also want to go beyond uniformly magnetized state and exploit the ability to continuously morph the equilibrium magnetic texture in the azimuthal direction as a means of engineering the selection rules and thus coherently access otherwise hidden mode symmetries.