Fast dual-beam alignment method for stimulated emission depletion microscopy using aggregation-induced emission dye resin

Miao Zhao; Fengming Liu; Yang Yu; Xinjun Guo; Hao Ruan; Jing Wen

doi:10.3788/COL202220.113601

Abstract

A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated emission. The coincidence degree of these two beams in three dimensions will significantly influence the spatial resolution of the microscope. However, the conventional alignment approach based on raster scanning of gold nanoparticles by the two laser beams separately suffers from a mismatch between fluorescence and scattering modes. To circumvent the above problems, we demonstrate a fast alignment design by scanning the second beam over the fabricated sample, which is made of aggregation-induced emission (AIE) dye resin. The relative positions of solid and donut laser beams can be represented by the fluorescent AIE from the labeled spots in the dye resin. This design achieves ultra-high resolutions of 22 nm in the x/y relative displacement and 27 nm in the z relative displacement for fast spatial matching of the two laser beams. This study has potential applications in scenarios that require the spatial matching of multiple laser beams, and the field of views of different objectives, for example, in a microscope with high precision.