Super-resolved microscopy by temporal encoding of single-molecule localization
Dirigée par Sandrine Lévêque-Fort
Fluorescence microscopy is widely used to study biological systems by selectively imaging fluorescent molecules. Among super-resolution techniques, single-molecule localization microscopy (SMLM) determines the position of individual emitters with nanometric precision from diffraction-limited images, typically acquired with scientific cameras. While this approach has proven highly successful, camera-based detection remains limited by frame rate and temporal integration, restricting the observation of fast dynamic processes.
This thesis presents Time Localization Microscopy (TimeLoc), a localization approach in which spatial information is encoded in the temporal modulation of the fluorescence signal rather than in its spatial distribution. A translating interference pattern modulates the fluorescence emitted by each molecule at a position-dependent frequency, allowing its position to be estimated from the detected photon stream using frequency analysis.
The work includes the theoretical description of the method, the development of a dedicated experimental microscope integrating both single-SPAD and 23-pixel SPAD-array detection, and the data-analysis framework required to recover molecular positions from photon-counting measurements. The experimental implementation is validated using fluorescent beads, showing good agreement with the theoretical localization performance. Different excitation strategies for one- and two-dimensional localization are investigated, and a frequency-to-position calibration procedure is established for quantitative localization. The capabilities of TimeLoc are further demonstrated through single-particle tracking and multi-emitter localization. Finally, preliminary dSTORM and DNA-PAINT experiments on biological samples provide an initial assessment of TimeLoc for super-resolution imaging.