Diversification of the 10th core atom of 9-cyanopyronins expands the resonance Raman vibrational palette

9-Cyanopyronin derivatives have been used as resonance Raman probes for highly sensitive and multiplexed vibrational biological imaging. Here, we synthesized a series of fourteen 9-cyanopyronin derivatives in which the 10th core atom is substituted with various group 13, 14, 15 or 16 elements, and systematically evaluated their properties using electronic pre-resonance stimulated Raman scattering (EPR-SRS) microscopy. Substitution at the 10th position modulates not only the vibrational frequency of the nitrile (C ≡ N) group in the silent region, but also the conjugated double-bond stretching frequency (C = C) in the fingerprint region, and additionally alters the subcellular localization of the probes. By employing different combinations of probes, we were able to utilize the distinct patterns of C ≡ N or C = C bond vibrations together with the different subcellular localizations of the probes to achieve live-cell three-color imaging. These 9-cyanopyronin derivatives greatly expand the vibrational palette available for multiplexed SRS imaging. Rhodamine derivatives with different core atoms at the 10th position exhibit diverse optical properties for fluorescence imaging, however, the effect of the 10th core atom on vibrational properties remain largely underexplored. Here, the authors prepare 9-cyanopyronin derivatives in which the 10th core atom is substituted with group 13, 14, 15 or 16 elements and evaluate their properties as resonance Raman probes, performing live-cell three-color imaging using distinct patterns of C ≡ N or C = C bond vibrations together with different subcellular probe localization.