A154 - Adaptable excitation field for enhanced single-molecule regime in dSTORM

Lancelot Pincet (lancelot.pincet@universite-paris-saclay.fr)
Sandrine Lévêque-fort (sandrine.leveque-fort@u-psud.fr)

In Single Molecule Localization Microscopy (SMLM), the quality of the super-resolved image highly depends on the capacity to generate single molecule imaging conditions. dSTORM in particular strongly relies on the control of the fluorescent dye photophysics. A method, called ASTER (Adaptable Scanning for Tunable Emission Regions) has been recently proposed to generate an effective uniform excitation by introducing a fast scanning (kHz) of the gaussian LASER beam which creates dynamically a top hat excitation. We will see how this technology can achieve wide filed of views (150x150µm²) with several sectioning angles (EPI, HILO, TIRF). We will then present how ASTER can be upgraded into IASTER (Intelligent ASTER) by coupling a dynamic zoom based on focal-tunable lenses. By simultaneously changing the width of the beam and its position, we can create an intelligent excitation pattern that would adapt to the imaged sample. Indeed, high labelling density is a known limitation that can deteriorate the single molecule regime. By previously studying the dye photoswitching response, we can efficiently adapt the scanning pattern of IASTER in these regions. This offers a smart way to redistribute the excitation photons in order to counteract high localizations density. We will show how this technique can be used in time, during an SMLM acquisition, to trigger an optimized and uniform dye photoswitching in the sample. We will finally show how the acquisition improvements can be quantified with Localisation density map, FRC, and SQUIRREL measurements. Acquisitions will be made on homemade 1 color – 2D samples (ex. Microtubules), but other samples from the participants would be welcome.