Synergic Combination of Stimulated Emission Depletion Microscopy with Image Scanning Microscopy to Reduce Light Dosage

Abstract

Stimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing the STED beam intensity, it theoretically improves the spatial resolution to any desired value. However, the higher is the dose of stimulating photons, the stronger are the photo-bleaching and photo-toxicity effects, which potentially compromise live-cell and long-term imaging. For this reason the scientific community is looking for strategies to reduce the STED beam intensity needed to achieve a target resolution. Here, we show how the combination of STED microscopy with image scanning microscopy (ISM) meets this request. In particular, we introduce a new STED-ISM architecture - based on our recent single-photon-avalanche-diode (SPAD) detector array - which allows covering the near-diffraction limit resolution range with reduced STED beam intensity. We demonstrate this ability both with simulated data and in live-cell experiments. Because of (i) the minimal changes in the optical architecture of the typical point-scanning STED microscope; (ii) the parameter-free, robust and real-time pixel-reassignment method to obtain the STED-ISM image; (iii) the compatibility with all the recent progresses in STED microscopy, we envisage a natural and rapid upgrade of any STED microscope to the proposed STED-ISM architecture.