Multiplexed intravascular molecular imaging using dual-channel near-infrared fluorescence integrated with optical coherence tomography

Abstract Accurate characterization of high-risk atherosclerotic plaques requires both structural and molecular information. While integrated optical coherence tomography (OCT)/near-infrared fluorescence (NIRF) imaging enables simultaneous assessment of plaque morphology and biological activity, single-target molecular detection may be insufficient to characterize the multifactorial plaque progression. Multiplexed intravascular molecular imaging remains technically challenging due to fluorescence crosstalk between emission channels, which compromises signal specificity. Here, we present a temporally modulated dual-channel NIRF/OCT catheter system designed to suppress spectral crosstalk and enable simultaneous multiplexed molecular imaging with structural information within the vessel wall. Time-interleaved excitation synchronized to the OCT trigger generates two NIRF excitation signals with a half-cycle phase difference, enabling crosstalk-free detection of two distinct molecular targets, as verified by phantom experiments. In vivo imaging in a rabbit model of atherosclerosis, using probes targeting calcified regions associated with osteogenic activity (ALN-PEG-Cy5.5) and mannose receptor (MMR)-expressing cells, including specific macrophage subset (MMR-PEG-Cy7), enabled simultaneous visualization of distinct molecular signatures within OCT-defined plaque structures. Ex vivo fluorescence imaging and immunohistochemistry supported the biological relevance of each NIRF channel. This integrated multimodal catheter system advances intravascular imaging from single-marker detection toward multiplexed molecular assessment of plaques, which could enable a more comprehensive evaluation of plaque pathobiology.