Upconversion Super-resolution Microscopy
Photon upconversion offers an exciting opportunity for biological super-resolution imaging. By harnessing excitation and emission processes of upconversion, we discovered powerful nonlinear depletion and emission, enabling subcellular super-resolution microscopy by breaking the unbroken traditional limitations. Stimulated emission depletion (STED) microscopy has become a powerful diffraction-unlimited technique for fluorescence imaging with resolution governed by d=λ/(2nsinθ(1+I/Isat)1/2). However, STED fundamentally suffers from high-intensity light illumination, photobleaching, re-excitation background, sophisticated probe-defined laser schemes, and limited photon budget of the probes. In 2015, we realized emission depletion in UCNPs. In 2017, using UCNPs we developed low-power CW laser enabled nonbleaching cytoskeleton STED imaging. In our very recent progress, we have successfully broken the theoretical limit of saturation intensity itself by two orders using new mechanism, and provides background-free, contrast-enhanced imaging at λex/38 resolution. We demonstrate a versatile strategy, stimulated-emission induced excitation depletion (STExD), to deplete the emission of multi-chromatic probes using a single pair of low-power, NIR CW lasers with fixed wavelength. With the effect of cascade amplified depletion effects, we achieve emission depletion for a wide range of emitters by manipulating their common sensitizer. We demonstrate an ultrahigh depletion efficiency of 99.3% for the 450 nm emission with a record low saturation intensity of 23.8 kW cm-2. We further demonstrate nanoscopic imaging with a series of multi-chromatic nanoprobes, two-color STExD imaging, and subcellular imaging of the immunolabelled actin filaments.
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South China Normal University
P. R. China