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

Significance: 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.

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

Approach: 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.

Results: 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.

Conclusions: 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.