Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette

Abstract

Vibrational imaging such as Raman microscopy is a powerful technique for visualizing a variety of molecules in live cells and tissues with chemical contrast. Going beyond the conventional label-free modality, recent advance of coupling alkyne vibrational tags with stimulated Raman scattering microscopy paves the way for imaging a wide spectrum of alkyne-labeled small biomolecules with superb sensitivity, specificity, resolution, biocompatibility, and minimal perturbation. Unfortunately, the currently available alkyne tag only processes a single vibrational “color”, which prohibits multiplex chemical imaging of small molecules in a way that is being routinely practiced in fluorescence microscopy. Herein we develop a three-color vibrational palette of alkyne tags using a 13C-based isotopic editing strategy. We first synthesized 13C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theoretical predictions, the mono-13C (13C≡12C) and bis-13C (13C≡13C) labeled alkyne isotopologues display Raman peaks that are red-shifted and spectrally resolved from the originally unlabeled (12C≡12C) alkynyl probe. We further demonstrated three-color chemical imaging of nascent DNA, RNA, and newly uptaken fatty-acid in live mammalian cells with a simultaneous treatment of three different isotopically edited alkynyl metabolic reporters. The alkyne vibrational palette presented here thus opens up multicolor imaging of small biomolecules, enlightening a new dimension of chemical imaging.

Super-multiplex vibrational imaging

“…As indicated as z-axis, combinatorial isotopic editing of the two atoms involved in the triple bond (Fig. 3a, highlighted in red and blue) enables local modulation of their reduced mass into 4 possible combinations 24 that further expand the vibrational palette.…”

The chemistry of the carbon-transition metal double and triple bond: Annual survey covering the year 2014

“…of alkynyl nucleosides (e.g. 1554) [1322] and for the synthesis of BODIPY dimers (e.g. 1557) (also via olefin metathesis) [1323].…”

Live-cell bioorthogonal Raman imaging

“…The vibration wavenumber of a Raman reporter is affected by the neighboring functional groups [14 ]. Furthermore, isotopic substitution, for example, 13 C substitution in alkynes, may result in shifted bioorthogonal Raman reporters that are spectrally resolvable [15 ]. Taken together, multi-color imaging using bioorthognal Raman reporters may be readily performed.…”