Alkyne Tags for Multiplexed, Optical Photothermal Infrared Imaging of Cellular Metabolites

Optical photothermal infrared (OPTIR) microscopy has emerged as a powerful strategy for vibrational imaging within complex cellular environments. This platform offers subcellular resolution without the diffraction limits of traditional IR. However, the potential for high-level multiplexing has been slowed by a lack of optimized, IR-active bioorthogonal probes. Building on our previous success with sulfur-linked alkyne tags for Raman imaging, we report a panel of compact, sulfur-linked IR-vibrational probes. These motifs feature strategic chemical modifications to maximize n-π* and d-π interactions, effectively activating the infrared transition dipole of the alkyne moiety. We validated the tools by labeling model lipid targets in cells, demonstrating that the probes exhibit robust, spectrally resolvable signals. This new library of bioorthogonal tags significantly broadens the analytical capabilities of OPTIR, providing a scalable framework for highly multiplexed, high-sensitivity metabolic imaging.