Tracking down fluorophores to the angstrom level by MINSTED.

Abstract

The ability to localize a fluorophore down to its physical size is a unique feature of state of the art nanoscopy methods like MINFLUX and MINSTED. MINSTED achieves this localization precision with minimal number of detected photons by stimulated emission depletion (STED) and detection-driven repositioning of the central minimum of the STED donut towards the fluorophore position. STED, as one of the key principles of MINSTED, makes use of a dedicated light pattern to inhibit the fluorophore's ability to fluoresce. The intensity needed is proportional to the stimulated emission cross-section of the fluorophore at the laser wavelength. This wavelength is a compromise between cross-section and direct excitation of the fluorophore. Conventional STED imaging cannot benefit much from shifting the STED wavelength closer to the emission maximum, as peripheral fluorophores are excited by the STED laser. By contrast, MINSTED, operating on singularized emitters, can make use of STED light at shorter wavelength to localize emitters down to the angstrom level. Using switchable fluorophores, this allows imaging of both reference DNA origami samples and biological specimens, with localization precisions smaller than the physical size of the fluorophore. Such high localization precisions pave the way for new insights into biological systems, as the instrument is not the resolution-limiting factor anymore.