Visualization of purine and pyrimidine de novo synthesis and salvage pathway activity in single-cell using Carbon Isotope Imaging and Spectral Tracing (CIIST)
Nitrogenous bases, namely purine and pyrimidine, and their derivatives are key metabolites for the growth and division of cells as they are involved in the storage of genetic information, protein synthesis, energy carrier molecules, and many other metabolic processes. Here we report a single-cell Raman imaging technique for nitrogenous base pathway mapping in prokaryotic and eukaryotic microbial systems via carbon isotope imaging and spectral tracing (CIIST). This method helps in visualizing the turnover dynamics of de novo synthesized purine and pyrimidine nitrogenous bases at the sub-cellular level over time. The enrichment of carbon isotope (carbon-12 or carbon-13) in the nitrogenous base generates Raman peaks at different positions. CIIST can also help in visualizing the salvage pathway activity by identifying new exogenously transported purine and pyrimidine into the cell. CIIST can also be used for generating spatial maps for quasi-quantitative imaging of nitrogenous base turnover in cells. Overall findings provides support for the prospective utility of CIIST technique as a highly effective tool for spatiotemporal and multiplex biomolecular analysis of nitrogenous base metabolism with unmatched spatial resolution in a single cell.