Vibrational tag coupled Raman imaging for visualizing biosynthesis pathways in action

Single cells are complex dynamic system which requires advanced multiplexing tools for imaging of biomolecules, their synthesis and turnover. Raman Imaging is a non-destructive vibrational imaging technique for spatio-temporal distribution and visualisation of multiple biomolecules such as amino acids, nucleic acids, proteins, lipids and others simultaneously. In our study, we have used stable isotopes as vibrational tags which show a distinct shifted peak assigned to biomolecules in Raman spectra when compared to their unlabelled counterpart. We have grown carbon-13 labelled microbial cells in the culture medium with an unlabelled (carbon- 12) carbon source and acquired the Raman image of a single microbial cell at different time points. The singlecell Raman image in the early hour shows a highly intense signal distribution from the carbon-13 labelled biomolecule peak position whereas a diminished signal intensity from the unlabelled biomolecular peak position. As incubation time progresses, image intensity distribution from the carbon-13 labelled peak weakens and the nascent unlabelled peak becomes more pronounced. Our finding generates spatial maps to visualize the turnover distribution of newly synthesized biomolecules giving insight into biomolecular dynamics and biosynthesis pathways in action. The vibrational tag Raman imaging technique has the potential for understanding and examining nascent biomolecular dynamics in situ and in vivo.