Sub-60-nm isotropic 3D super-resolution microscopy through self-interference field excitation

Due to its unique optical sectioning capability, confocal laser scanning microscopy (CLSM) can provide highly sensitive, highly specific imaging of specimens in three dimensions and has been recognized as an indispensable tool for biological and medical studies. Nonetheless, the spatial resolution of CLSM is constrained by the diffraction nature, with λ /2 resolution laterally ( x y ) and 1.5 λ resolution axially ( z ). To improve the imaging resolution beyond the diffraction limit as well as to achieve its isotropy, we present a strategy of mirror-assisted self-interference field excitation (SIEx) highly nonlinear microscopy. The imaging principle has been theoretically modeled and investigated in accordance with the Wolf vector diffraction theory. The experimental demonstration of isotropic three-dimensional SIEx nanoscopy, assisted with the ultrahigh-order optical nonlinearity of photon avalanching nanoparticles, was achieved utilizing a common laser-scanning microscope configuration, resulting in a lateral resolution of 54 nm ( λ /15) and an axial resolution of 57 nm ( λ /15) with one single beam from a low-power, continuous-wave, near-infrared laser (19kW⋅cm −2 ). We further extended the applicability of the SIEx scheme to biological imaging and demonstrated super-resolution imaging for immunolabeled actin filaments of BSC-1 cells with an isotropic full width at half maximum of ∼67nm ( λ /13). Our facile SIEx methodology can, in principle, be seamlessly integrated with the existing and widely available laser-scanning fluorescence microscopes without adding any complexity, thereby enabling their capability of 3D isotropic super-resolution imaging.