Deep imaging by NIR-II multifocal structured illumination microscopy with enhanced super-resolution radial fluctuation

Compared to wide-field (WF) microscopy in the second near-infrared (NIR-II) window, NIR-II multifocal structured illumination microscopy (NIR-II MSIM) provides a twofold improvement in transverse spatial resolution, along with enhanced penetration depth and superior image contrast. These advantages position NIR-II MSIM as a promising platform for studying physiological processes in turbid specimens. However, significant background noise caused by cumulative photon scattering continues to severely degrade image resolution and fidelity. Moreover, the data processing workflow in NIR-II MSIM is highly sensitive to motion artifacts, which further limits its broader applications in live-tissue imaging. To address these challenges, we introduce NIR-II MSIM-eSRRF, a technique that combines NIR-II MSIM with enhanced super-resolution radial fluctuation (eSRRF). This hybrid approach facilitates advanced deep imaging with improved spatial resolution and contrast across turbid specimens, including Intralipid phantoms, ex-vivo pork tissue, and live mice. Notably, NIR-II MSIM-eSRRF could effectively suppress motion artifacts, yielding nearly artifact-free visualization of fine cerebral vessels in vivo. As such, the NIR-II MSIM-eSRRF platform will emerge as a powerful tool for investigating physiological processes in highly scattering environments, thereby accelerating advancements in intravital fluorescence microscopy.