Multifocal metalenses drive super-resolution imaging of brain organoids

Attaining sub-diffraction-limit resolution in thick, scattered biological tissues remains a pivotal challenge in optical microscopy. In a recent publication in Light: Science & Applications, Jo et al. presented a notable advancement: a hybrid-multiplexed multifocal metalens engineered for image scanning microscopy (ISM) that achieved high-resolution, deep-tissue imaging of brain organoids within a compact optical system (Fig. 1)¹.

Fig. 1  Illustration of the multifocal metalens (left) and pixel reassignment reconstruction (right).

ISM improves spatial resolution by replacing the single-point detector of traditional confocal systems with an array detector, allowing fine sampling of individual excitation spots. Through pixel reassignment and subsequent deconvolution, ISM can realise up to a two-fold enhancement in resolution beyond the diffraction limit^2,3. To boost temporal resolution, multifocal illumination schemes are utilised to parallelise the scanning process^4,5, making ISM ideal for high-speed and volumetric imaging in biological applications. Numerous strategies have been developed to generate multifocal excitation, including digital micromirror devices (DMDs) and microlens arrays (MLAs). However, these approaches face limitations due to trade-offs between cost, numerical aperture (NA), pitch, and multifocal uniformity. Metalenses—ultrathin nanostructured surfaces capable of precise phase modulation—present a promising alternative. Their design flexibility and compact form factor complement traditional methods such as MLAs, pinhole arrays, beam-splitting prisms, spatial light modulators (SLMs), DMDs, and diffractive optical elements (DOEs). Table 1 offers a comparative summary of various multifocal generation techniques.

To overcome the drawbacks of conventional methods, Jo et al. developed a hybrid multiplexing technique that combines two existing approaches: phase addition and random multiplexing¹. By splitting the desired focal pattern into interleaved even and odd subarrays, random multiplexing was independently applied using distinct random binary masks to reduce interference between neighbouring foci. The resulting phase profiles were then merged through simple addition, enabling the formation of a dense and well-isolated multifocal pattern. This method generates a 40 × 40 focal array with a 3-µm pitch and 0.7 NA, fabricated on a silicon nitride platform optimised for 488-nm excitation.

To demonstrate the practical application of their design, the authors integrated a metalens into a custom-built ISM platform termed multifocal metalens-based ISM (MMISM), which they used to image fluorescently labelled human forebrain organoids with up to 40 µm thickness. The system achieves neuronal feature resolution of approximately 330–370 nm—nearly twice that of widefield microscopy—while effectively suppressing out-of-focus background through the digital pinholing process.

Building on the principle that orthogonally polarised light beams do not interfere, Jo et al. extended their method to polarisation hybrid multiplexing, enabling the generation of denser and more uniform focal arrays¹. This innovation not only increases focal density without inducing crosstalk but also reduces the number of required scanning frames, facilitating faster imaging. Additionally, the technique holds promise for future applications in chiral-selective imaging, where polarisation is crucial.

Combined with scalable fabrication methods like nanoimprint lithography and adaptability to multiphoton excitation for deeper imaging, the approach maps a clear path toward practical, high-throughput 3D imaging in organoid and tissue systems. Jo et al. emphasised that their hybrid multiplexing strategy extends beyond multifocal metalenses, offering a versatile framework for integrating arbitrary phase profiles in multifunctional metalens designs¹. This flexibility allows multiple optical functions to be incorporated into a single compact device, simplifying system architecture. Recent advancements, such as the s²ISM introduced by Zunino et al.¹², which computationally removes out-of-focus signals, and the C²SD-ISM developed by Liang et al.¹³, which physically eliminates the defocused background using a spinning disk, have maintained the super-resolution capabilities of ISM while significantly enhancing optical sectioning in thick or scattering samples. MMISM could significantly enhance its imaging performance by integrating these cutting-edge techniques.