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

Phototrophs assimilate CO₂ into organic compounds that accumulate in storage organelles. Elucidation of the carbon dynamics of storage organelles could enhance the production efficiency of valuable compounds and facilitate the screening of strains with high photosynthetic activity. To comprehensively elucidate the carbon dynamics of these organelles, the intraorganellar distribution of the carbon atoms that accumulate at specific time periods should be probed. In this study, the biosynthesis of polysaccharides in storage organelles was spatiotemporally probed via stimulated Raman scattering (SRS) microscopy using a stable isotope (¹³C) as the tracking probe. Paramylon granules (a storage organelle of β-1,3-glucan) accumulated in a unicellular photosynthetic alga, Euglena gracilis, were investigated as a model organelle. The carbon source of the culture medium was switched from NaH¹²CO₃ to NaH¹³CO₃ during the production of the paramylon granules; this resulted in the distribution of the ¹²C and ¹³C constituents in the granules, so that the biosynthetic process could be tracked. Taking advantage of high-resolution SRS imaging and label switching, the localization of the ¹²C and ¹³C constituents inside a single paramylon granule could be visualized in three dimensions, thus revealing the growth process of paramylon granules. We propose that this method can be used for comprehensive elucidation of the dynamic activities of storage organelles.