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

Figure. 1 | Working principle of the multifocal metalens. a, An illustration of the multifocal metalens. b, an illustration of the MMISM setup. MM, multifocal metalens; IP, intermediate image plane; BE, beam expander; DM, dichroic mirror; BP, bandpass filter; TL, tube lens; obj., objective lens. c, schematics of hybrid multiplexing methods and representative simulated PSFs of multifocal arrays generated using hybrid multiplexing. The multifocal arrays were designed with parameters of 20 × 20 foci, 3 μm pitch, 0.7 NA at a wavelength of 488 nm. Scale bar: 10 AU. @ Yongjea Jo et al.

ISM is a super-resolution imaging technique that enhances resolution beyond the diffraction limit through pixel reassignment and deconvolution. However, the practical implementation of ISM requires the generation of dense and uniform multifocal arrays. Conventional optical modulators, such as digital micromirror devices and microlens arrays, have been used to generate multifocal arrays, but they suffer from complex alignment, limited numerical aperture (NA), and long optical paths, resulting in bulky and inefficient systems.

In a new paper published in Light: Science & Applications, a research team led by Professor Inki Kim at the BioNanoPhotonics Laboratory in the Department of Biophysics, Institute of Quantum Biophysics, Sungkyunkwan University, developed a multifocal metalens based on a novel hybrid multiplexing approach to implement ISM and successfully captured super-resolution images of neuronal structures in brain organoids. The proposed hybrid multiplexing method combines two conventional approaches, phase addition and random multiplexing, to generate a highly uniform and dense multifocal array by leveraging the strengths of both methods. This innovation allows for the creation of multifocal arrays with higher NA and smaller pitch than conventional multifocal metalens designs.

Using this approach, the team designed a metalens with NA 0.7, capable of generating a 40 × 40 focal arrays. The metalens was fabricated via electron beam lithography, and its optical performance was experimentally validated. The measured point spread functions (PSFs) showed excellent agreement with simulation results. Furthermore, the team integrated the metalens into a custom-built multifocal metalens-based ISM (MMISM), which successfully demonstrated super-resolution imaging of brain organoids. The system achieved a two-fold improvement in optical resolution compared to WF imaging and clearly resolved fine neuronal fibers that cannot be resolved using WF systems. Furthermore, digital pinholing method effectively suppressed background noise in thick tissue samples, enabling high-contrast, optically sectioned images.

In addition, the team introduced a polarization-based hybrid multiplexing design, in which right- and left-handed circularly polarized (RCP/LCP) light are independently controlled using Pancharatnam–Berry phase design approach. By interleaving RCP and LCP focal arrays, the pitch can be further reduced without introducing interference between foci, resulting in a higher density mutifocal array. Simulation results confirmed that this polarization hybrid design achieves superior uniformity even at reduced pitch sizes compared to hybrid multiplexing without utilizing PB phase.

At the core of the MMISM system is a multifunctional metalens—an ultrathin, flat optical component designed with the hybrid multiplexing method to generate dense and uniform multifocal arrays. The system can be further optimized through polarization control, enabling even tighter focal spacing while maintaining high resolution and imaging quality.

The researchers describe the working principle of their metalens as follows:

"The hybrid multiplexing method combines the advantages of conventional multiplexing techniques to generate a denser and more uniform focal array by minimizing interference between neighboring focal spots."

The team also explained their work as follows:

“In neuroscience, observing fine structures in deep, scattering tissue is crucial to understanding brain function. MMISM provides approximately twice the resolution of WF microscopy and reveals sharper neuronal fiber details through improved resolution and optical sectioning," they added.

“The hybrid multiplexing strategy offers a flexible design platform that can be applied not only to multifocal metalenses but also to various multifunctional metalens applications. It enables the integration of multiple optical elements into a single ultrathin metalens, paving the way for simpler and more compact imaging systems. These advances could lead to the next generation of high-performance optical microscopy systems.” the scientists forecast.

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

Yongjae Jo, Hyemi Park, Seho Lee, Hyeyoung Yoon, Taehoon Lee, Gyusoo Bak, Hanjun Cho, Jong-Chan Park & Inki Kim

https://www.nature.com/articles/s41377-025-01900-3

Chinese Academy of Sciences