Streamlining the cell flow: Feasibility of acoustically driven cell alignment for in vivo flow cytometry

In vivo flow cytometry (IVFC) utilizes blood vessels as natural conduits for real-time and noninvasive monitoring of circulating cells. However, conventional IVFC systems are primarily limited to superficial vessels, restricting analytical throughput and diagnostic sensitivity. Here, we propose a novel acoustic-based cell alignment strategy that allows IVFC to be applied in a broader range of vascular locations. We developed a dual ultrasound transducer (DUST) system in which two transducers are positioned face-to-face at the same angle. This configuration generates an interference-based acoustic field containing periodically arranged pressure nodes and antinodes within the vessel. The resulting field aligns flowing cells into multiple parallel streamlines, concentrating their movement within a confined region and enhancing the consistency and efficiency of signal detection. Blood vessel mimicking phantom experiments demonstrated that a dual ultrasound (DUS) enables stable multiple parallel streamlines of microbeads in a vessel while maintaining uniform flow velocity. Furthermore, fluorescent beads modeling rare cells exhibited approximately a 9-fold increase in signal-to-noise ratio (SNR) under DUS application compared to the non-aligned condition. Signal intensity fluctuations at the detection point were also significantly reduced, enabling more stable and reliable signal analysis. This approach demonstrates strong potential for highly sensitive, single-cell-level diagnostics in vivo. It also enables seamless integration with photoacoustic or fluorescence-based detection systems for future multimodal single-cell analysis.