Multiplexed optoacoustic tracking and magnetic actuation of labeled blood cells in living mice

Visualization of cellular dynamics in microvascular networks is essential for deciphering physiology and disease. Existing imaging platforms commonly lack the spatiotemporal resolution and sensitivity for single-cell tracking in vivo, particularly beyond the penetration depth of optical microscopy. While recent fluorescence and optoacoustic methods allow detection of single microparticles in vivo, multiplexed imaging of distinct circulating cell populations remains unachieved. Here, we present an optimized labeling protocol using near-infrared (NIR) dyes ICG (indocyanine green) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) to generate spectrally distinct optoacoustic signatures for red blood cells (RBCs) and neutrophils. This enables noninvasive simultaneous tracking of both cell types in deep cerebrovascular networks of mice, supporting superresolution imaging with localization optoacoustic tomography and revealing differences in the velocities of different cell types inside opaque tissues. ICG-labeled RBCs are further detectable via diffuse optical localization imaging in the NIR-II window, permitting high-resolution visualization of cortical capillaries. In addition, incorporation of superparamagnetic nanoparticles allows noninvasive magnetic manipulation of the microcirculation alongside real-time single-cell monitoring, further providing a strategy for precise and reversible vascular occlusion in preclinical ischemic stroke models. Together, these capabilities provide a versatile platform for advancing vascular research at the single-cell level.