Two-photon excitation STED microscopy

We report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with stimulated-emission depletion (STED). We demonstrate an easy-to-implement and promising laser combination based on a short-pulse laser source for two-photon excitation and a continuous-wave (CW) laser source for resolution enhancement. Images of fluorescent nanoparticles and the immunostained transcription regulator NF kappa B in mammalian cell nuclei exhibit resolutions of <50 nm and similar to 70 nm in the focal plane, respectively, corresponding to a 4-5.4-fold improvement over the diffraction barrier.