Fig. 2. The silicon rhodamine dyes ONB-2SiR, HCage 620 and pPA-SiR can be efficiently photoactivated by a two-photon process with 515 nm light.

a Chemical structures of ONB-2SiR, HCage 620 and pPA-SiR before (upper row) and after activation (lower row). b Absorption spectrum of the unactivated caged form (dark gray), and absorption and emission spectra of the activated uncaged form (light gray, red) for ONB-2SiR. The green and blue lines centered at 515 nm and 375 nm indicate the laser light employed for the 2PA and 1PA, respectively. Exc., STED: excitation and STED wavelengths. c Experimental sequence: Frame 0: reference scan with the excitation beam alone to detect residual background fluorescence of unactivated dye; Activation: scan with 515-nm femtosecond laser to uncage (activate) fluorophores; Frame 1: readout of the fluorescence signal with the excitation beam. Below: the images of tubulin before and after activation (10 × 10 µm², 256 × 256 pxl²). Scale bar: 10 µm. All beams were diffraction-limited, the activation and readout sequence were repeated 39 times. d Average fluorescence signal vs. number of consecutive readout frames after activation scans for different activation powers P_act. For low activation power, the signal initially increases. Later, photobleaching is dominant, resulting in a signal decrease. e A 3-level system, which takes the activation and bleaching process into account, is sufficient to describe the observed signal behavior (curves in d). f Activation and bleaching rate extracted from fits to the data in d as a function of activation power. The power scaling indicates a two-photon process for activation, and more complex photobleaching behavior¹⁸. Error bars represent standard error of the mean (s.e.m.), which is similar to the marker size in many instances. Source data are provided as a Source Data file.