245 / 2024-08-21 11:20:17

Three-dimensional super-resolution microscopy by employing mirror-assisted single-beam interference

Super-resolution microscopy,isotropic imaging,axial resolution,multiphoton fluorescence,photon avalanching

Three-dimensional super-resolution microscopy is a powerful tool for information technologies as well as life sciences, but usually with the axial resolution distinctly worse than the lateral one because only a fraction of the uniform spherical wavefront can be collected by an objective lens. To achieve isotropic super resolution imaging, we present a conceptually novel strategy of mirror-assisted self-interference field excitation highly-nonlinear microscopy. We demonstrated isotropic three-dimensional imaging assisted with the ultrahigh-order (33^rd) optical nonlinearity of photon avalanching nanoparticles based on a common laser-scanning microscope configuration, achieving a lateral resolution down to 54 nm (λ/15) and an axial resolution down to 57 nm (λ/15) with one low-power CW beam (19 kW·cm^-2). By introducing a spatial light modulator optically conjugated to the rear focal plane of the objective lens, bifocal vector field modulation can be achieved, and the mirror-assisted single-beam strategy can realize a single-objective 4Pi illumination. Furthermore, ultrahighly nonlinear excitation can completely eliminate the intrinsic sidelobes in the 4Pi focus with no need of any mathematical deconvolution postprocessing, resulting in a sidelobe-free extremely narrow fluorescence excitation spot with an axial resolution down to 26 nm (λ/33). The facile single-objective, single-beam strategy is highly cost-effective, robust, stable and easy to implement in terms of optical architectures. It can be readily compatible with well-commercialized raster or parallel laser-scanning microscopy, thereby advancing cutting-edge technologies such as super-resolution imaging, laser lithography, and optical data storage.