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.

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Fig: Spectroscopy principle and probe design of electronic pre-resonance SRS microscopy: a Schematic illustration of electronic pre-resonance SRS. Two synchronized laser beams were tightly focused on molecules of interest. When the energy difference between two photons matches vibrational transition, one pump photon can be converted to stokes photon and the intensity fluctuation will be detected. As pump energy approaches electronic transition (pre-resonance), such process will be greatly enhanced. ω0ωpump, and ωvib represents for frequencies of electronic transition, pump beam photon, and vibrational transition, respectively. Γ is the homogeneous linewidth, typically around 700 cm−1. b Design and engineering principles of 9-cyanopyronin library. Three key structural features were rationally tuned to generate new MARS dye library. One sidechain was installed on the amino group to facilitate facile functionalization.

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.

Miao, Y., Qian, N., Shi, L. et al. 9-Cyanopyronin probe palette for super-multiplexed vibrational imaging. Nat Commun 12, 4518 (2021). https://doi.org/10.1038/s41467-021-24855-6

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