Quad-Channel In Vivo Photoacoustic Multiplexing Using Tunable Gold Nanorods.

Photoacoustic (PA) imaging combines the molecular specificity of optical absorption with the resolution and depth of ultrasound, enabling noninvasive molecular imaging in vivo. However, robust and accurate PA multiplexing is hindered by the broad spectra and poor photostability of existing contrast agents, and interference from endogenous absorbers. Here, we demonstrate four-channel PA multiplexing using PEGylated silica-coated gold nanorods with narrow, spectrally distinct plasmon resonances spanning the near-infrared window. A reproducible three-step seed-mediated synthesis yields monodisperse, spectrally tunable gold nanorods. Co-hybrid silica encapsulation with PEGylation enhances photostability and standardizes both particle dimensions and surface chemistries across the panel. With only a commercial OPO laser and a straightforward non-negative least-squares algorithm, we achieve accurate signal separation across a full-factorial set of in vitro and in vivo models, maintaining mean absolute errors below 4.3%. We further show that the nanorod library remains clearly distinguishable in the presence of endogenous oxyhemoglobin and deoxyhemoglobin and can be unmixed using VisualSonics native spectral deconvolution. This platform doubles existing PA multiplexing capabilities to four exogenous channels without requiring multimodal imaging, complex laser setups, or complicated computational pipelines. This work establishes a framework for future applications in noninvasive biomarker mapping, multicellular therapies, and spatially resolved diagnostics.