Abstract
Context and Objective: Autotrophic microalgae can produce a number of substances such as polysaccharides, lipids, proteins, carotenoids, polyphosphates, or crystalline purines directly from inorganic sources. Different microalgal species are used for the economic production of high-value products. Due to their autotrophic nature, microalgae can biosynthesize complex isotopically labeled biomolecules from simple isotopically labeled inorganic substances. Analysis of the chemical composition of microalgae by means of chemical-analytical methods is relatively complex, time-consuming, and laborious. Confocal Raman microscopy (CRM) represents practical optical method by which the biosynthesis of isotopically labeled compounds can be monitored in situ, i.e. directly within intact cells.
Methods: The CRM combining confocal optical microscopy with Raman spectroscopy enables fast and non-destructive analysis of the chemical composition of substances in the investigated objects, including the effect of isotopic labeling. The chemical composition of the investigated objects is reflected by their Raman spectra, in the case of Raman mapping of microscopic objects by their chemical maps. In this work, two specific cases of isotopic labeling were studied, namely the effect of heavy water (deuteration) and 15N-enrichment of biomolecules in living cells of the marine dinoflagellate Amphidinium carterae.
Results and Conclusions: The main result was the detection of variously deuterated and 15N-enriched forms of crystalline guanine depending on the ratio of D2O/H2O and 15N/14N-labelled nitrates in the culture medium and the determination of the mean degree isotopic labeling by comparison of the biosynthetized guanine with synthetically prepared isotopic forms of guanine. An interesting and valuable discovery, which was not originally the intention of this work, was the finding of unexpected persistence against the dissolution of nanocrystalline guanine even in highly diluted solutions.