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Elimination of Re‐excitation in Stimulated Emission Depletion Nanoscopy Based on Photon Extraction in a Phasor Plot

Stimulated emission depletion (STED) nanoscopy can achieve super‐resolution imaging of microstructures at the nanoscale by confining the fluorescence emission of a diffraction‐limited focusing spot. The depletion wavelength is typically at the red tail of the emission spectrum of the fluorophore to avoid re‐excitation; however, inefficient fluorescence inhibition on account of the small stimulated emission cross‐section results in a huge demand for the depletion of laser power. Here, an accurate photon extraction method is proposed to eliminate re‐excitation caused by blue shift of the depletion wavelength. After only extracting the photons unaffected by a donut‐shaped depletion laser in the phasor plot, both the resolution and signal‐to‐noise ratio of the STED images are significantly improved. As a result, the increased depletion efficiency at a blue shifted depletion wavelength reduces the power demand for achieving a certain resolution. This approach, with flexible wavelength selectivity owing to the independence of depletion power, made more fluorophores applicable for STED super‐resolution imaging, thereby offering more convenient and efficient service to researchers.