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 H 2 O 2 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/H 2 O 2 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.