Dynamic Nonlinear Stepwise Optical Saturation for Super‐Resolution Imaging with Upconversion Nanoparticles

Abstract Upconversion nanoparticles (UCNPs) exhibit exceptional nonlinear optical properties that offer powerful capabilities for super‐resolution imaging. In this study, we exploit the dynamic nonlinear characteristics of lanthanide‐doped UCNPs to propose a multi‐order dynamic nonlinear stepwise optical saturation (DN‐SOS) image scanning microscopy (ISM), effectively overcoming the diffraction limit inherent in conventional ISM techniques. To elucidate the nonlinear fluorescence behavior of UCNPs under varying excitation conditions, a complete rate‐equation model is established, and Taylor expansion of the fluorescence signal reveals that higher‐order terms encode high spatial frequency information for resolution enhancement. By dynamically modulating excitation power during image acquisition, nonlinear fluorescence response images are captured, and a multi‐image weighted finite difference method is applied to suppress lower‐order components in the Taylor expansion, thereby extracting high spatial frequency details. Although unlimited resolution enhancement is theoretically attainable, practical performance is constrained by the signal‐to‐noise ratio (SNR). Utilizing a custom‐built confocal setup with 980 nm excitation laser and 800 nm detection, we demonstrate fourth‐order DN‐SOS imaging with a lateral resolution of ≈130 nm, around an eighth of the excitation wavelength. This corresponds to a threefold improvement beyond the diffraction limit. The method offers a simple solution for super‐resolution imaging in point‐scanning systems without complex synchronization schemes.