Image scanning microscopy based on multifocal metalens for sub-diffraction-limited imaging of brain organoids

Image scanning microscopy (ISM) is a promising imaging technique that offers sub-diffraction-limited resolution and optical sectioning. Theoretically, ISM can improve the optical resolution by a factor of two through pixel reassignment and deconvolution. Multifocal array illumination and scanning have been widely adopted to implement ISM because of their simplicity. Conventionally, digital micromirror devices (DMDs)¹ and microlens arrays (MLAs)^2,3 have been used to generate dense and uniform multifocal arrays for ISM, which are critical for achieving fast imaging and high-quality ISM reconstruction. However, these approaches have limitations in terms of cost, numerical aperture (NA), pitch, and uniformity, making it challenging to create dense and high-quality multifocal arrays at high NA. To overcome these limitations, we introduced a novel multifocal metalens design strategy called the hybrid multiplexing method, which combines two conventional multiplexing approaches: phase addition and random multiplexing. Through numerical simulations, we demonstrate that the proposed method generates more uniform and denser multifocal arrays than conventional methods, even at small pitches. As a proof of concept, we fabricated a multifocal metalens generating 40 × 40 array of foci with a 3 μm pitch and NA of 0.7 operating at a wavelength of 488 nm and then constructed the multifocal metalens-based ISM (MMISM). We demonstrated that MMISM successfully resolved sub-diffraction-limited features in imaging of microbead samples and forebrain organoid sections. The results showed that MMISM imaging achieved twice the diffraction-limited resolution and revealed clearer structural features of neurons compared to wide-field images. We anticipate that our novel design strategy can be widely applied to produce multifunctional optical elements and replace conventional optical elements in specialized applications.