Index matching improves the imaging quality of 3D well-of-the-well dishes for embryo culture

The success rate of in vitro fertilization (IVF) is low ( $<33\%$ ), driving the need for techniques to improve embryo quality. One promising solution is the well-of-the-well (WOW) culture system. WOWs with 3D microwells offer superior culture quality over 2D microwells; however, the refractive index (RI) mismatch between the 3D microwells and culture media causes aberrations that limit imaging quality and hinder clinical translation.

To enable clinical deployment of WOW dishes, we demonstrate the fabrication of index-matching 3D WOW dishes that improve embryo imaging quality.

We applied 3D printing and molding to fabricate agarose-based WOW dishes. The fabricated product was inspected using optical coherence tomography, and its reproducibility was validated. We applied a slanted-edge method to characterize optical resolution through substrates with different RIs and assessed geometric distortion of WOW dishes with different RIs using certified microspheres. To evaluate additional RI-dependent optical aberrations, we performed Shack-Hartmann wavefront sensing and Zernike analysis on WOW dishes of different RIs. Finally, we assessed the stability of agarose microwells after a 6-day incubation and demonstrated in situ imaging using mouse embryos.

We confirmed that the proposed fabrication method has acceptable reproducibility. We found that low-RI material mitigates spherical aberration, improves optical resolution compared with high-RI materials, reduces geometric distortion, and eliminates visibility of ridge structures. Consistent with these observations, wavefront analysis showed that high-RI materials generated substantially higher-order aberrations, whereas low-RI materials produced minimal higher-order error. We validated that the agarose microwells retained their shapes after a 6-day incubation. We confirmed normal development of mouse embryos over 4 days in agarose-based dishes, indicating agarose-based WOW dishes are feasible for embryo culture.

The proposed fabrication strategy eliminates a barrier to the integration of 3D WOW dishes for both culture and selection and may enable their clinical use to enhance IVF success rates.