9-Cyanopyronin probe palette for super-multiplexed vibrational imaging

Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities. The authors develop a method to build Manhattan Raman Scattering (MARS) probes based on different core atoms, conjugation ring numbers, and stable isotope substitutions. A quantitative model predicts vibrational frequencies of MARS dyes from structures, which are used in supermultiplexed vibrational imaging.