Stimulated emission depletion (STED) nanoscopy enables the visualization of subcellular organelles in unprecedented detail. However, reducing the power dependency remains one of the greatest challenges for STED imaging in living cells. Here, we propose a new method, called modulated STED, to reduce the demand for depletion power in STED imaging by modulating the information from the temporal and spatial domains. In this approach, an excitation pulse is followed by a depletion pulse with a longer delay; therefore, the fluorescence decay curve contains both confocal and STED photons in a laser pulse period. With time-resolved detection, we can remove residual diffraction-limited signals pixel by pixel from STED photons by taking the weighted difference of the depleted photons. Finally, fluorescence emission in the periphery of an excitation spot is further inhibited through spatial modulation of fluorescent signals, which replaced the increase of the depletion power in conventional STED. We demonstrate that the modulated STED method can achieve a resolution of < 100 nm in both fixed and living cells with a depletion power that is dozens of times lower than that of conventional STED, therefore, it is very suitable for long-term super-resolution imaging of living cells. Furthermore, the idea of the method could open up a new avenue to the implementation of other experiments, such as light-sheet imaging, multicolor and three-demensional (3D) super-resolution imaging.

Many kinds of nanoparticles and organic dyes as fluorescent probes have been used in the stimulated emission depletion (STED) nanoscopy. Due to high toxicity, photobleaching and non-water solubility, these fluorescent probes are hard to apply in living cell imaging. Here, we report a new fluorescence carbon dots (FNCDs) with high photoluminescence quantum yield (56%), low toxicity, anti-photobleaching and good water-solubility that suitable for live-cell imaging can be obtained by doping fluorine element. Moreover, the FNCDs can stain the nucleolus and tunneling nanotubes (TNTs) in the living cell. More importantly, for STED nanoscopy imaging, the FNCDs effectively depleted background signals and improved imaging resolution. Furthermore, the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained. And because of their good water dispersibility, the higher resolution of single FNCDs size in the nucleolus of a living cell can be up to 19.7 nm. After the image optimization steps, the fine fluorescence images of TNTs diameter with ca. 75 nm resolution is obtained living cell, yielding a threefold enhancement compared with that in confocal imaging. Additionally, the FNCDs show excellent photobleaching resistance after 1, 000 scan cycles in the STED model. All results show that FNCDs have significant potential for application in STED nanoscopy.