Raman microscopy shows that nitrogen-rich cellular inclusions in microalgae are microcrystalline guanine

•Raman microscopy was used for in situ identification of cytoplasmic inclusions.•Inclusions in two microalgal species were identified as crystalline guanine.•The crystalline guanine was suggested to serve as a compact depot of nitrogen.•Raman microscopy can discriminate between inclusions of different composition.

Microalgal cells possess a vast diversity of subcellular structures and cytoplasmic inclusions differing in their morphology, functionality, and composition, some of them giving rise to distinct Raman spectral signatures allowing their identification, localization, and visualization in situ. Here, we show that certain Raman features observed in Raman spectra of microalgae can be unambiguously attributed to guanine microcrystals because they are clearly distinct from Raman fingerprints of closely related purine species. Using confocal Raman microscopy, we have localized crystalline guanine as a part of cellular inclusions in the chlorophyte Desmodesmus quadricauda and in the eustigmatophyte Trachydiscus minutus. We propose that this finding also explains the chemical nature of similar nitrogen-rich crystalline structures recently documented in a number of other chlorophyte species by energy-dispersive X-ray spectroscopy. To the best of our knowledge, this is the first Raman microscopy-based direct evidence of the presence of guanine microcrystalline inclusions within microalgal cells. We tentatively propose that the crystalline guanine serves as a very compact, long-term depot of nitrogen in microalgae. Simplicity of specimen preparation requiring no fixation, labeling, or staining of the cells predetermines Raman microscopy as a method of choice for more advanced studies of the physiological role of guanine particles, as well as other crystalline inclusions in situ within intact cells.

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