Versatile Ultrasound-Compatible Microfluidic Platform for In Vitro Microvasculature Flow Research and Imaging Optimization

Ultrasound localization microscopy (ULM) enables the creation of super-resolved images and velocity maps by localizing and tracking microbubble contrast agents through a vascular network over thousands of frames of ultrafast plane wave images. However, a significant challenge lies in developing ultrasound-compatible microvasculature phantoms to investigate microbubble flow and distribution in controlled environments. In this study, we introduce a new class of gelatin-based microfluidic-inspired phantoms uniquely tailored for ULM studies. These devices allow for the creation of complex and reproducible microvascular networks featuring channel diameters as small as 100 μm. Our experiments focused on microbubble behavior under ULM conditions within bifurcating and converging vessel phantoms. We evaluated the impact of bifurcation angles (25, 45, and 55°) and flow rates (0.01, 0.02, and 0.03 mL/min) on the acquisition time of branching channels. Additionally, we explored the saturation time effect of narrow channels branching off larger ones. Significantly longer acquisition times were observed for the narrow vessels, with an average increase of 72% when a 100 μm channel branched off from a 300 μm channel and an average increase of 90% for a 200 μm channel branching off from a 500 μm channel. The robustness of our fabrication method is demonstrated through the creation of two trifurcating microfluidic phantoms, including one that converges back into a single channel, a configuration that cannot be achieved through traditional methods. This new class of ULM phantoms serves as a versatile platform for noninvasively studying complex flow patterns using ultrasound imaging, unlocking new possibilities for in vitro microvasculature research and imaging optimization.