Projects

In neuroscience, often the ultimate goal is to reveal the organization of the circuits that correlate and explain animal behavior and brain disease. Nevertheless, one of the biggest challenges to measuring (multiple) neuronal circuits is when they are located in deep brain regions only accessible to optical endoscopic approaches. Among them, methods based on thin multimode fibers (MMFs) are promising tools for measuring neuronal activity in deep brain regions of freely moving mice thanks to their small diameter. However, current techniques are limited: while fiber photometry approaches provide only an ensemble integrated signal of neuronal activity, imaging techniques that can measure single-cell signals use long multimode fibers, which are very sensitive to bending and have not been applied to unrestrained rodents yet. Here, we demonstrated the fundamentals of a new approach using a short MMF coupled to a miniscope. In proof-of-principle in vitro experiments, we disentangled spatio-temporal fluorescence signals from multiple fluorescent sources transmitted by a thin (200 µm) and short (8 mm) MMF, using a general unconstrained non-negative matrix factorization algorithm directly on the raw video data. Furthermore, we show that low-cost open-source miniscopes have sufficient sensitivity to image the same fluorescence patterns seen in our proof-of-principle experiment, suggesting a new avenue for novel minimally invasive deep brain studies using multimode fibers in freely behaving mice. 

The cellular shape (morphology) and motility are properties closely related to the cell's function and behavior in a variety of physiological processes including cell division, cell migration and tissue shape changes. A better understanding of how cells move and change their shape will help us not only elucidate how these physiological processes occur but also what goes awry in disease, for example during the uncontrolled growth and metastasis of cancer cells. Animal cell morphology and movement are provided by cytoskeletal fibers, in particular actin filaments. Actin filaments are abundant and directly responsible for most shape changes known in cells: phagocytosis, cytokinesis, cell migration, cell contraction (muscles), etc. Actin filaments are made of globular actin molecules whose polymerization is tightly controlled in space and time. The versatility of actin assembly geometries (single filaments, bundles, 2D networks, and 3D gels) and their ability to constitute mechanosensitive structures (sarcomeres, stress fibers, and adherens junctions) make actin filaments the major molecular target for studying cell morphology and motility. However, many key questions on how cells control actin filament assembly and organization remain unclear, particularly at the nanoscale. The main reason why there are so many unanswered questions on how cells assemble and organize cytoskeleton proteins is the lack of suitable techniques to probe nanometric scale cellular structures in a noninvasive manner. Nevertheless, using polar-(d)STORM, a novel polarized fluorescence superresolution microscopy, our group was able for the first time to disentangle the structural disorganization of the target molecule (actins) from the fluorophore floppiness, and therefore perform nanoscale structural imaging. However, due to some limitations, the technique is not applicable to any complex biomolecular structure of interest. Within this context, this PhD project aims at developing a novel polarized super-resolution microscopy, the 4polar-dSTORM, and to use this novel technique to unveil how cells organize and assemble actin filaments at the nanoscale. In particular, compared ensemble measurements of polar-spinning disk microscopy to the novel single-molecule and super-resolution fluorescence polarized microscopy approach (4polar-dSTORM) and realized that actin filament alignment is quite robust against different contractility conditions.

The increasing microorganism resistance to antibiotics is a matter of global concern and it is evident that research on new antimicrobial molecules and on new strategies are crucial. In particular, the interaction studies of antimicrobial molecules of which act on the membranes of bacteria are promising candidates. Among these, macromolecules (polymers) have many advantages when compared to other small biocides: they have increased lifetimes, potency and specificity and lowered residual toxicity. Besides, antimicrobial polymers can be functionalized, some of them are amphiphiles (they self-assemble into nanoscale aggregates in liquids) and a few have good optical properties for photodynamic action. In other words, there is a great technological potential behind of antimicrobial polymers - from the antimicrobial coating of solid surfaces to antimicrobial fibers for many scientific or biomedical applications such as sterile clothing and bandages. The present research has as a goal the investigation of the molecular mechanism of which Cationic Polyelectrolytes (CP) interact with a biomimetic membrane model (Langmuir Film made of lipids). For that, we will use the SFG Spectroscopy - the Surface Nonlinear Vibrational Spectroscopy by Sum-Frequency Generation, which is an intrinsically surface-specific technique. The SFG signal does not have the contribution from the molecules in solution (centrosymmetric media). Therefore, by using SFG Spectroscopy it allows us to obtain information about the orientation and the molecular conformation at the interface. For example, if the polyelectrolyte adsorbs without any preferential orientation (a disordered coiled conformation for example), no SFG signal would be detected. In the case of lipids, the technique can easily detect if their hydrophobic chains are extended or have gauche defects (folding in the chain). In addition, by its vibrational spectrum, one could determine whether the lipid has been oxidized. Therefore, SFG Spectroscopy is perfect for analyzing the interaction between the lipid monolayer on the water (of which simulates half of the cell membrane) and the CP, since it obtains information at the molecular level. The understanding of these interactions may elucidate the detailed mechanism of action of antimicrobial CP,  allowing that new molecules could be planned in order to optimize their bactericidal activity or biocompatibility.