Live-Cell Multiplexed Imaging and Chemical Sensing with Cumulene and Polyyne Allotropes

Visualizing a large number of species in living systems is essential for understanding complex life processes. With unique vibrational spectroscopy, polyyne has demonstrated multiplexed Raman imaging through fine-tuned frequencies in the cell-silent window (1800-2700 cm^-1). Here, we develop new polyyne allotropes for multiplexed imaging and vibrational sensing in live cells by stimulated Raman scattering (SRS) microscopy. Cumulenes are engineered to obtain a vibrational palette with 5 distinct frequencies in the range of 1900-2050 cm^-1, achieving previously inaccessible frequencies for SRS imaging. Ratiometric polyyne sensors are further developed to demonstrate multiplexed Raman sensing of both γ-glutamyl transpeptidase (GGT) and H₂O₂ reactive species in live cells. By combining cumulenes and polyynes, 10-color optical imaging and chemical sensing have been achieved in living cells to visualize both organelle interactions and changes of endogenous GGT/H₂O₂ levels under drug treatment. The development of multiplexed imaging and functional sensing with polyyne allotropes shows great potential for studying subcellular activities and interactions in live cells.