Abstract
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.
Highlights
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
9cyano xanthene was selected as the parent structure to generate Manhattan Raman Scattering (MARS) dye library for electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy
Major advances in optical imaging have been largely driven by spectroscopy and imaging probes
Summary
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. 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). Sequential labeling and imaging, such as cyclic immunofluorescence and DNA exchangebased multiplexing (Exchange-PAINT), was developed to expand the multiplexing capacity[11,12,13,14], these methods cannot work with live samples and they are clearly time- and labor-consuming. When the pump laser energy approaches fluorophores’ electronic transition but remains detuned from the rigorous resonance, the vibrational signal from the fluorophore backbone can be enhanced by >104 through the vibrational and electronic coupling[20] Harnessing this electronic pre-resonance effect with SRS microscopy leads to a desirable combination of high detection sensitivity Epr-SRS microscopy of MARS probes has emerged as an ultrasensitive method to image specific biological targets inside cells with vibrational contrast
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