Higher-Order Nonsaturated Hyperlinear Upconversion Image Scanning Microscopy for Super-Resolution Imaging

Conventional confocal laser scanning microscopy (CLSM) requires a trade-off between image quality and resolution. Image scanning microscopy (ISM) uses an array detector to improve this issue, but its diffraction-limited resolution improvement is capped at twice that of CLSM. Here, we demonstrate a novel lanthanide-doped upconversion nanoparticles (UCNPs) based super-resolution approach, termed nonsaturated hyperlinear upconversion image scanning microscopy (UISM). By modulating the excitation power of UCNPs during image acquisition, the hyperlinear response of UCNPs can be tuned at the nonsaturated excitation threshold to obtain a shrunken point spread function, enabling resolution enhancement far beyond twice that of CLSM. Utilizing nonsaturating near-infrared excitation at 980 nm, we achieve spatial resolutions of 133 and 196 nm for emissions at 455 and 800 nm, respectively, corresponding to approximately 1/5th and 1/8th of the excitation wavelength. This work suggests a novel strategy for improving image resolution in laser point-scanning microscopy.