Bleaching protection and axial sectioning in fluorescence nanoscopy through two-photon activation at 515 nm

Activation of caged fluorophores in microscopy has mostly relied on the absorption of a single ultraviolet (UV) photon of ≲400 nm wavelength or on the simultaneous absorption of two near-infrared (NIR) photons >700 nm. Here, we show that two green photons (515 nm) can substitute for a single photon (~260 nm) to activate popular silicon-rhodamine (Si-R) dyes. Activation in the green range eliminates the chromatic aberrations that plague activation by UV or NIR light. Thus, in confocal fluorescence microscopy, the activation focal volume can be matched with that of confocal detection. Besides, detrimental losses of UV and NIR light in the optical system are avoided. We apply two-photon activation (2PA) of three Si-R dyes in different superresolution approaches. STED microscopy of thick samples is improved through optical sectioning and photobleaching reduced by confining active fluorophores to a thin layer. 2PA of individualized fluorophores enables MINSTED nanoscopy with nanometer-resolution.