Multiplexed Shortwave Infrared Imaging Highlights Anatomical Structures in Mice

Multiplexed fluorescence in vivo imaging remains challenging due to the attenuation and scattering of visible and traditional near infrared (NIR‐I, 650–950 nm) wavelengths. Fluorescence imaging using shortwave infrared (SWIR, 1000–1700 nm, a.k.a. NIR‐II) light enables deeper tissue penetration due to reduced tissue scattering as well as minimal background autofluorescence. SWIR‐emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability are powerful contrast agents, yet few imaging demonstrations exclusively use SWIR emission beyond two‐color imaging schemes. In this study, we engineered three high quality lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emission peaks ranging from 1100–1550 nm for simultaneous three‐color imaging in mice. We first use the exceptional photostability of QDs to non‐invasively track lymphatic drainage with longitudinal imaging, highlighting the detailed networks of lymphatic vessels with widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature networks, including clearly distinguishing the liver and spleen. This work establishes optimized SWIR QDs for next generation multiplexed and longitudinal preclinical imaging, unlocking numerous opportunities for preclinical studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms. We synthesized three high quality lead sulfide/cadmium sulfide (PbS/CdS) quantum dots (QDs) across shortwave infrared (SWIR, 1000–1700 nm) imaging window for in vivo anatomical imaging. With a single color, we visualized tortuous network of lymphatic vessels and semi‐quantitatively tracked lymphatic drainage over time. Combining all three QD colors, we demonstrated multiplexed imaging to highlight overlapping vasculature and lymphatic structures with singular 808 nm excitation.