Ultralow power demand in fluorescence nanoscopy with digitally enhanced stimulated emission depletion

Stimulated emission depletion (STED) microscopy breaks the optical diffraction barrier and has become a powerful tool for biological study. However, its application for in vivo study is limited because of its high demand for depletion power. Here, we propose digitally enhanced STED (DE-STED) as a method for reducing the depletion power that is required for STED superresolution imaging. A donut image is the key in this approach, which is composed of the depleted photons by STED laser and represents the intensity difference between confocal and STED images from the same imaging position. The depletion efficiency is digitally enhanced by multiplying the intensity of the donut image with a factor greater than 1, and then the photons from the periphery of the diffraction-limited spot are fully depleted by subtracting the enhanced donut image from the original confocal image. Finally, DE-STED achieves a resolution of ~λ/8 in biological samples with a depletion power that is an order of magnitude lower than that in traditional STED imaging. Furthermore, the proposed method helps to relax the restrictions on the fluorophore because of its low phototoxicity and photobleaching.