9-Cyanopyronin probe palette for super-multiplexed vibrational imaging

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.

Bringing Vibrational Imaging to Chemical Biology with Molecular Probes

“…Furthermore, by harnessing the narrow line width of Raman peaks (50–100 times narrower than fluorescence peaks), researchers have developed highly sensitive Raman probe palettes. The matching dye palettes enable super-multiplexed (more than 20 channels) optical imaging for organelles or protein profiling with sensitivity down to 250 nM, bridging optical imaging’s subcellular spatial resolution with system biology’s high information throughput. − Moreover, chemically activatable and photochromic SRS probes have also been developed lately for intracellular sensing and multiplexed tracking, empowering functional SRS imaging. − …”

Miniaturized Chemical Tags for Optical Imaging

“…Further multiplexed capabilities have been achieved by modification of the xanthene core through ring expansion, replacement of the central atoms and isotopic editing of the appended alkyne and nitrile groups (Figure 6). [96] One of the first applications of small molecule ratiometric Raman sensing demonstrated intracellular pH measurement using the pH-sensitive C≡N stretching frequency of carbonylcyanide ptrifluoromethoxyphenylhydrazone. [97] Building from this concept, the group of Tomkinson synthesized a family of alkyne sensors for intracellular pH sensing, spanning a broad range of pH values, between 2 and 10.…”

“…Reproduced with permission. [96] Copyright 2021 Springer Nature under a creative commons license. C) Ratiometric SRS imaging of mitochondrial pH using Mitokyne.…”

Super‐Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy

“…[ 6,9 ] Owing to this drastic enhancement, we achieved nanomolar sensitivity of Raman‐active dyes (such as commercial far‐red fluorescent dyes and specially‐designed manhattan raman scattering (MARS) probes), and demonstrated epr‐SRS imaging of specific proteins. [ 6,10,11 ]…”

“…[6,9] Owing to this drastic enhancement, we achieved nanomolar sensitivity of Raman-active dyes (such as commercial far-red fluorescent dyes and specially-designed manhattan raman scattering (MARS) probes), and demonstrated epr-SRS imaging of specific proteins. [6,10,11] Collectively, SRS imaging circumvents the fundamental bottlenecks of fluorescence microscopy and provides unique benefits including label-free chemical imaging of endogenous biomolecules, [1,12] nonperturbative mapping of small metabolites, [4] and simultaneous profiling of a large group of specific biomarkers. [6,11] Yet, being an optical technique that uses near-infrared or visible light, SRS microscopy is still constrained to diffraction-limited spatial resolution (≈300 nm).…”