Full-field single molecule localization microscopy with a mono-detector.

Abstract

Fluorescence super-resolution microscopy has reached the ultimate detection limit of individual molecules. The sparsity property has enabled a change of paradigm from a diffraction-limited resolution in the few hundred nanometer range to a molecular localization-based resolution in the few nanometer range. Usually, single molecule localization microscopies are based on PSF fitting using a camera detector. Alternatively, another localization strategy is possible based on the dependence of the fluorophore emission on the illumination. A sequence of acquisition using different structured illuminations can be used to localize the emitter. This excitation-based strategy has been applied successfully for wide-field images using a shifting modulated illumination synchronized with a camera (ModLoc, SIMFlux, ROSE,…) and in a scanning approach configuration (MinFlux) with a single detector. However, these localization techniques are limited by the slowness of the camera in their full-field configuration and by the scanning approach in their mono-detector configuration. In this work, we present a novel concept for molecular localization microscopy where the information of the entire full-field image is given by the emitted photon flux. Although working as a full-field technique, this allows emitter localization via the acquisition of the fluorescence light by a single photon counting module, rather than a camera, which has the immediate advantage of gaining temporal resolution. The theoretical resolution limit (given by its Cramér-Rao lower bounds) gives a better localization precision as compared to the standard PSF fitting. We will show experimental characterization of this new localization technique in terms of spatial precision and temporal performances. Imaging of calibrated samples, as well as cytoskeleton network in COS7 cells will be presented, and the different assets of this new approach will be fully discussed.