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Alkynes were employed as tags to observe small molecules in cells by Raman microscopy. Herein, simple deuteration was found to shift the vibrational frequency of the alkyne by 135 cm−1. Two-color Raman imaging of D-alkynes and H-alkynes made it possible to distinguish between and observe similar small molecules in live cells.

Graphical abstract: Deuteration of terminal alkynes realizes simultaneous live cell Raman imaging of similar alkyne-tagged biomolecules

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