Sub-40 nm resolution deep tissue imaging by image scanning emission saturation nanoscopy
The development of deep-tissue super-resolution imaging serves as an essential bridge toward non-invasive in vivo optical observation. However, there remain challenges to balance spatial resolution, imaging depth and phototoxicity. Here, we present a nanoscopy namely Image Scanning Emission Saturation (ISES) nanoscopy, achieving a lateral resolution of 37 nm, 1/25th of the excitation wavelength, at an imaging depth of 200 {\mu}m. Using a 976-nm doughnut-shaped excitation beam within an imaging-scanning microscopy configuration, we apply saturation-based point spread function (PSF) engineering and pixel-level confocal-pinhole enhancement to improve spatial resolution. As the high- and low-frequency components of the image OTF are concurrently acquired in a single scan via different camera pixels, Fourier-domain fusion can be employed with a single scanning dataset to further improve image quality. Compared with the traditional doughnut excitation beam-based adaptive pixel reassignment method, our strategy preserves the original frequency distributions and mitigates reconstruction artifacts in complex biological sample imaging. This strategy is generalizable and compatible with a variety of probes displaying saturation behavior. Beyond enabling a versatile and practical approach for deep tissue super-resolution imaging, it also informs the development of next-generation nanoprobes for imaging.