Charitra Sree Senthil Kumar will defend her thesis entitled “Polarization-Resolved Single Molecule and Anisotropy Imaging of Actin Architecture and T-Cell Receptor Dynamics” on January 30, 2026 at 09:30 a.m. in room Pierre Cotton at Institut Fresnel (level -1), campus St Jérôme, Marseille.
Composition of the Jury :
– Dr Bassam HAJJ, Institut Curie, CNRS, Paris, Reviewer
– Prof. Steven F. LEE, Yusuf Hamied Department of Chemistry, University of Cambridge, UK, Reviewer
– Dr Ignacio IZEDDIN, Institut Langevin, Université PSL, Paris, Examiner
– Prof. Sandrine LÉVÊQUE-FORT, Institut des Sciences Moléculaires d’Orsay (ISMO), CNRS, Paris-Saclay, President of the Jury
– Prof. Sophie BRASSELET, Institut Fresnel, CNRS, Marseille, PhD Supervisor
– Prof. Hai-Tao HE, Centre d’Immunologie de Marseille-Luminy (CIML), CNRS, Marseille, Co-supervisor
Abstract : Single-Molecule Localization Microscopy (SMLM) has revolutionized nanoscale biological imaging with imaging resolutions reaching Angstrom scales. However, electron microscopy remains unparalleled for its ability to reveal true orientations in the sample, at the cost of tedious sample preparation, involving clearing out most cellular contents. This could lead to missing key information of the dynamics of intracellular processes that rely on both spatial arrangement and orientation of biomolecules with each other. Hence, live cell access to molecular orientation is crucial for understanding signaling processes. Single Molecule Orientation and Localization Microscopy (SMOLM) addresses this by determining both the position and orientation of fluorescent molecules, which are intrinsically encoded in their point spread function (PSF). PSF engineering and fitting techniques have been developed to extract this information, but they often involve complex setups, an enlarged PSF size and computationally intensive analyses, limiting their applicability in dense biological environments.
Building on the previously developed 4Polar2D technique which retrieves 2D orientation and wobbling of fluorescent molecules through ratiometric analysis across four polarization channels with minimal PSF deformation, this thesis presents 4Polar3D, an extended approach that recovers full 3D molecular orientation. The method introduces a simple back focal plane intensity filter to distinguish off-plane tilt angles, enabling robust 3D orientation retrieval with minimal effects on PSF size or shape. Using 4Polar3D, I image 3D actin architectures in dense cellular systems, including lamellipodia in tumor cells, 3D podosomes in macrophages, and the T-cell cytoskeleton on non-activating and activating planar substrates.
Beyond orientation imaging, I exploit the combination of 4Polar3D with controlled excitation polarization to quantify molecular rotational mobility at the single-molecule level, which is highly dependent on the fluorophore’s nature and environment. Finally, I investigate the ensemble actin organization at early signaling complexes formed after T-cell activation at physiological immunological synapses. Through fluorescence anisotropy measurements, I probe the rotational dynamics and molecular packing by assessing potential homoFRET within activated TCR-CD3 complexes.
Keywords : Polarization, Optical super-resolution microscopy, Actin organisation, T-Cell receptor, Fluorescence Anisotropy
