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Abstract:
In stimulated emission depletion (STED) nanoscopy the
wavelength of the STED beam is usually tuned towards the red tail of the
emission maximum of the fluorophore. Shifting the STED wavelength
closer to the emission peak, i.e. towards the blue region, favorably increases
the stimulated emission cross-section. However, this blue-shifting also
increases the probability to excite fluorophores that have remained in their
ground state, compromising the image contrast. Here we present a method
to exploit the higher STED efficiency of blue-shifted STED beams while
maintaining the contrast in the image. The method is exemplified by
imaging immunolabeled features in mammalian cells with an up to 3-fold
increased STED efficiency compared to that encountered in standard STED
nanoscopy implementations.