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We developed a new class of two-photon excitation-stimulated emission depletion (2PE-STED) optical microscope. In this work, we show the opportunity to perform superresolved fluorescence imaging, exciting and stimulating the emission of a fluorophore by means of a single wavelength. We show that a widely used red-emitting fluorophore, ATTO647N, can be two-photon excited at a wavelength allowing both 2PE and STED using the very same laser source. This fact opens the possibility to perform 2PE microscopy at four to five times STED-improved resolution, while exploiting the intrinsic advantages of nonlinear excitation.

Original publication




Journal article


Proc Natl Acad Sci U S A

Publication Date





6390 - 6393