Fig. 1. Principle of alignment optical setup for dual-beam STED microscopy. (a) Schematic diagram of dual-beam adjustment optical path. (b) The writing process of the labeled spot by the excitation beam. (c) Distribution of each fluorescence intensity at the peak versus lateral and axial mismatches between the excitation beam and the inhibition beam.

Fig. 2. Fluorescent properties of AIE-induced dye resin sample with and without TPE. (a) Fluorescence spectra of dye resin sample with and without TPE, and both emission spectra are excited by the 370 nm laser. (b) Selection of AIE dyes with different inhibition beams.

Fig. 3. Labeled spot 3D feature size. (a) The variance of the lateral (x, y) size of the labeled spot versus the power of the 515 nm fs excitation laser. (b) SEM image of the labeled spot inside the dye resin sample with TPE. The lateral (x, y) size is 271 nm, and the axial (z) size is 992 nm.

Fig. 4. Variation of emission spectral intensity during the coarse pre-alignment process. (a) CCD real-time observation results of relative positions of two beams. (b) The change of fluorescence emission intensity with different relative positions of two beams.

Fig. 5. Measurement process of alignment resolution. Fluorescence emission intensity variance versus different relative positions of the two beams along (a) the lateral direction of x/y relative displacement and (c) the axial direction of the z relative displacement. (b), (d) Scale value of the staff gauge on the stage for controlling the movement of the beam and the corresponding physical displacement between the two beams Δx/Δz.