Focused ultrasound-based acoustic holography for 3D cell patterning and biomimetic microenvironment construction.

Focused Ultrasound (FUS) provides a non-contact, directional acoustic field extensively employed in diverse biomedical applications. However, conventional concave transducers are physically constrained to a single fixed focus, lacking the flexibility for dynamic field modulation necessary to meet increasingly complex functional requirements. In this work, we report a focused ultrasound-based acoustic holography (FUS-AH) approach that enables concave transducers to achieve sophisticated spatial control and generate intricate multi-focal patterns. By systematically investigating the propagation mechanics within the convergent zone, we discovered an intrinsic focused convergence-driven scaling mechanism that allows for continuous lateral zooming of complex acoustic fields along the Z-axis without altering the phase plate. Furthermore, the FUS-induced geometric convergence triggers self-interference of the modulated holographic field, yielding localized quasi-standing waves and enabling ultra-fine acoustic trapping in completely open, unconstrained environments, consistently reconstructing continuous complex topologies with sub-millimeter line widths (0.36-0.51 mm). Leveraging these capabilities, we demonstrated rapid, high-throughput, and cross-scale multilayer cell patterning, where cellular aggregates were successfully localized into highly ordered, uniform matrices with standardized cluster diameters (175.7-216.2 µm) and rigid geometric center-to-center pitches (coefficients of variation , CV ≤ 11.2%). Finally, a biomimetic multilayer tumor-vascular microenvironment model was established, exhibiting robust long-term structural maturation with a 74.47% tumor area expansion over a 12-day culture period. This technology holds substantial potential for applications in drug delivery, micro-manipulation, transcranial therapy, and tissue engineering.