Paralleled needle-like-beam image scanning microscopy for fast volumetric super-resolution imaging

Super-resolution (SR) fluorescence microscopy has proven to be an essential tool for investigating intricate biological systems. However, it frequently encounters challenges related to speed and complexity when conducting volumetric imaging of thick samples. Herein, we present an enhanced variant of parallelized image scanning microscopy (ISM) to address these challenges. By incorporating a pinhole in the illumination path to generate a needle-like beam that extends the depth of focus (DOF) of the focused beam array, the system enables synchronized capture of multifocal signals from eight distinct sample planes in combination with a multiplane prism. Notably, through improvements in the reconstruction algorithm, this method significantly reduces the required number of raw frames, shortening the SR imaging time for a volume of approximately 42 × 37 × 1.9 <i>μm</i>³ to sub-second-scale while maintaining axial imaging synchronization. This breakthrough enables the real-time SR volumetric imaging of thin biological samples, which we successfully demonstrated on live cell samples. Furthermore, we validated the rapid SR imaging capabilities of the system through experiments on various fluorescent samples, demonstrating its potential for transformative applications in biomedical imaging.