Low-power STED nanoscopy based on temporal and spatial modulation

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.