STED microscopy with a supercontinuum laser source

TLDR

The study presents a simple yet powerful STED microscopy implementation achieving far‑field subdiffraction resolution. The method uses a single super‑continuum pulsed laser for both excitation and depletion, simplifying pulse preparation and enabling multicolor imaging. At ~1 MHz repetition, the system reduces photobleaching and achieves 30–50 nm lateral resolution—an 8–9× improvement over the diffraction limit—demonstrated on dense nanoparticles and mammalian microtubules.

Abstract

We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same super-continuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier.