Research-SRS imaging of alkyne tags

Small biomolecules such as nucleosides, amino acids, choline, fatty acids and drugs are biologically vital species, playing indispensable roles inside cells. However, sensitive and specific visualization of these molecules in living systems has proven to be highly challenging. We report SRS imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals, by matching quantum amplification, bond-selectivity, background-free and biocompatibility of SRS with high polarizability, sharp Raman transition, spectral bioorthogonality and small size of C≡C moiety, respectively. We have demonstrated this technique by visualizing de novo synthesis of DNA, RNA, proteomes, phospholipids and triglycerides, respectively, through metabolic incorporation of alkyne-tagged small precursors (as shown in Fig. 1), and tracking alkyne-bearing drugs in mouse tissues. Our technique brings small biomolecules under the illumination of modern live-cell microscopy.


Fig. 1. SRS imaging of de novo synthesis of DNA, RNA, proteomes, phospholipids and triglycerides, through metabolic incorporation of alkyne-tagged deoxyribonucleoside (EdU), ribonucleoside (EU), amino acid (Hpg), choline (propargylcholine) and fatty acid (octadecynoic acid) into live cells.

Inspired by the color palettes of fluorescent tags, we attempted to expand the vibrational “colors” of alkyne tags to enable multi-chemical imaging. Realizing the vibrational frequency is related to the reduced mass of the oscillator, we synthesized EdU-13C (containing 13C≡12C) and EU-13C2 (containing 13C≡13C) with clearly resolved alkyne Raman peaks from the original 12C≡12C moiety. As shown in Fig. 2, three-color SRS imaging of 17-octadecynoic acid, EdU-13C and EU-13C2 thus demonstrates this isotopic editing strategy and paves the way for simultaneous visualization of multiple vibrational "colors".

Several ambitious biomedical applications are underway. One of them is to image the cell uptake dynamics of alkyne-tagged glucose, which could be a powerful method to probe cellular metabolic state. Another is to study neurogenesis by tracking the dynamics of proliferating neuronal stem cells (via DNA synthesis) in the hippocampus of brain tissues.


Fig. 2. Three-color SRS imaging of alkyne-tagged small metabolites: 17-octadecynoic acid (blue at 2125 cm-1), EdU-13C (green at 2077 cm-1) and EU-13C2 (red at 2048 cm-1).

Relevant Papers:
1. L. Wei, F. Hu, Y. Shen, Z. Chen, Y. Yu, C. Lin, M. Wang and W. Min. "Live-cell imaging with alkyne-tagged small biomolecules by stimluated Raman Scattering", Nat. Method (2014).[PDF]