Spatiotemporal monitoring of intracellular metabolic dynamics by resonance Raman microscopy with isotope labeling

Abstract

We probed production process of a cellular metabolite with a stable isotope-labeled substrate exposed to various conditions.

Probing the Biogenesis of Polysaccharide Granules in Algal Cells at Sub-Organellar Resolution via Raman Microscopy with Stable Isotope Labeling

“…This result indicates that the ratios of the 13 C and 12 C constituents of the granules corresponded to those of the carbon sources in the culture media owing to the fixations of 13 CO 2 and 12 CO 2 with equal probabilities, respectively. This result is similar to that of the resonance Raman spectral shifts of carotenoids, which were incubated with a 13 C source (β-carotene and astaxanthin in cyanobacterial and algal cells, respectively). Thereafter, these paramylon granules were visualized via SRS microscopy employing the spectra of 100% 13 C– and 100% 12 C–paramylon granules (Figure a), which were color-coded as red and green, respectively (Figure c).…”

“…Conversely, Raman microscopy combined with SI labeling can spatiotemporally probe metabolic dynamics, − thereby offering the following significant advantages for investigating storage organelles: first, cellular metabolic processes, even in a dynamic equilibrium, can be tracked by exposing cells to SI-labeled substrates, and the localization and distribution of the SI-labeled products in the cells can be detected by Raman microscopy . Second, SI-labeled substrates do not interfere with the biological functions of cells because their physiological properties are hardly affected by SI labeling .…”

“…Second, SI-labeled substrates do not interfere with the biological functions of cells because their physiological properties are hardly affected by SI labeling . Third, SI labeling is applicable to a wide range of target biomolecules, including proteins, − DNA, − lipids, − polysaccharides, , and small metabolites, − in different cell types by selecting SI-labeled substrates that are present in the desired metabolic pathways. Fourth, because Raman microscopy is free from photobleaching, Raman imaging combined with SI labeling can be used for long-term monitoring of organelle activity.…”

“…Conversely, Raman microscopy combined with SI labeling can spatiotemporally probe metabolic dynamics, 31−48 thereby offering the following significant advantages for investigating storage organelles: first, cellular metabolic processes, even in a dynamic equilibrium, can be tracked by exposing cells to SIlabeled substrates, and the localization and distribution of the SI-labeled products in the cells can be detected by Raman microscopy. 48 Second, SI-labeled substrates do not interfere with the biological functions of cells because their physiological properties are hardly affected by SI labeling. 49 Third, SI labeling is applicable to a wide range of target biomolecules, including proteins, 32−38 DNA, 37−39 lipids, 40−43 polysaccharides, 44,45 and small metabolites, 46−48 in different cell types by selecting SI-labeled substrates that are present in the desired metabolic pathways.…”

The Role of Raman Spectroscopy Within Quantitative Metabolomics

“…Shifts resulting from the incorporation of 13 C and 15 N atoms are mainly observed in the low wavenumber region of Raman spectra, and therefore the appearance of peaks in the silent region is not observed, as is seen from C-D in Raman-deuterium isotope probing (50). With a wide variety of possible isotopic substrates, Raman-stable isotope probing has been used to probe cell activity and the metabolism of a wide range of living organisms, including bacteria (42,48,51,(53)(54)(55)(56), algae (57), Caenorhabditis elegans (58), human cell lines (59), and human tissues (58).…”