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

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.