Versatile vasculature chips for ultrasound localization microscopy

Objective. Ultrasound localization microscopy (ULM) has revolutionized microvasculature imaging by surpassing the diffraction limit via microbubbles. While ULM demonstrates exceptional potential to resolve micron-scale vascular structures in both preclinical and clinical studies, its performance evaluation remains challenging primarily due to the lack of reference microvascular phantoms featuring realistic, micron-scale, and hierarchical vascular structures. This study thus aims to develop a fabrication protocol for microvasculature patterns that offers design versatility and enables on-demand customization. Approach. Inspired by microfluidic chip techniques, we present an organ-on-a-chip protocol for fabricating agarose-based micro-vessel network phantoms with ground truth. We experimentally demonstrated the feasibility of the vasculature phantom using two adapted patterns: (1) a leaf pattern, which exhibited intrinsic quasi-two-dimensional venation network with hierarchical and branching channels similar to animal vasculature, and (2) a kidney pattern derived from a two-dimensional projection of real human vasculature obtained via micro computed tomography. Microbubble solutions were perfused into the phantoms by capillary force and gravity. Main results. The ULM-reconstructed vasculature maps agreed well with the ground truth. ULM achieved high sensitivity values of 0.97 and 0.95, but low precision values of 0.37 and 0.60, for the leaf and kidney phantom, respectively. The results indicated the capability of ULM to reconstruct vessel structures while making many false positive predictions. Significance. The proposed protocol provides a versatile platform for creating realistic microvascular phantoms, facilitating the development, evaluation, and optimization of ultrasound microvascular imaging techniques.