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

Highlights

• 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.

Abstract

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.

Introduction

Microalgae use sunlight to produce depots of high free energy content such, as lipid, starch, or polyphosphate bodies, as well as to accumulate reserves of nutrients, such as carbon, phosphorus, sulfur, or nitrogen. Tapping these depots offers a great biotechnological potential [1], [2], [3]. Their assessment currently relies largely on extraction and chemical analysis of bulk algal biomass by gravimetry [4], [5], spectrophotometry and colorimetry [6], mass spectrometry [7], [8], gas and high-pressure liquid chromatography [8], [9], or nuclear magnetic resonance [9], [10]. On a more refined level, these methods can be targeted to a certain compound and/or cellular compartment to track down the origin of the investigated chemical within the cell [11], [12], [13].

Further improvement is available from methods allowing rapid, and simultaneous, identification and quantification of various biomolecules at the single-cell or even subcellular level. This is particularly important when working with heterogeneous cultures composed by cells in different physiological states and/or in different phases of their cell cycle. For imaging morphology and elemental/molecular composition of individual cells, microfluorescence [14], [15] and analytical electron microscopy [14] are the most exploited methods. Recently, analytical capabilities of (electron) microscopy complemented by energy-dispersive X-ray spectroscopy (EDX) [14], [16], secondary ion mass spectrometry (SIMS) [17], confocal Raman microscopy (micro-RS) [18], or combinations thereof, started to be used in algal studies because of their rich information content. Each method has its advantages, limitations, and information costs. Nanoscale SIMS (nano-SIMS) is an extremely sensitive mass spectrometric technique for determining the elemental, isotopic, or even molecular composition of a solid sample surface with a lateral resolution < 50 nm [19]. Similarly, analytical electron microscopy, including EDX, provides invaluable information on morphology and elemental composition. However, for imaging by nano-SIMS or EDX, the cells must be laboriously fixed, dried, and immobilized on a conducting surface or embedded in conducting resin. The complex sample preparation makes these low-throughput methods prone to artifacts arising from cell fixation, washing, staining and/or section preparation. Finally, elemental analysis per se is often insufficient for molecular identification.

Raman microscopy is a contactless, rapid, non-invasive, and often non-destructive method combining the advantage of molecular specificity of vibrational spectroscopy with a spatial resolution reaching the diffraction limit provided by confocal optical microscopy [20]. The application of micro-RS in biology is rapidly increasing [21] because it can provide biochemical and structural information without extensive sample preparation (e.g., fixation, staining, drying, cutting) and, in some cases, even on living cells [22]. Regarding microalgae, micro-RS was recently shown to be particularly interesting for the simultaneous detection and imaging of dense sub-cellular structures with a relatively homogenous chemical composition, such as starch grains, lipid bodies, or polyphosphate (polyP) granules [18], avoiding their staining or extraction.

In a similar way, confocal micro-RS might be useful for in situ express identification and imaging of crystal-like inclusions, profiting from the high local concentration and highly ordered structure of the molecular species of interest in such crystalline inclusions resulting in strong, sharp, and distinct Raman fingerprints facilitating spectral assignment. The crystal-like structures suitable for identification with micro-RS are exemplified by the vacuolar inclusions in a symbiotic dinoflagellate, Symbiodinium microadriaticum, which were long misidentified as calcium oxalate [23]. Only recently, using advanced imaging and analytical techniques (energy-filtered TEM analysis and mass spectrometry), these crystals have been shown to be composed of uric acid [24]. The microcrystals laboriously isolated from the cells of a free-living dinoflagellate Gonyaulax polyedra have been identified as guanine [25] according to the ultraviolet absorption spectra of their solution in hydrochloric acid, while vacuolar crystal-like particles found in calcareous dinophyte cells have been assumed to be calcite based simply on their solubility in acids [26]. In spite of a long research history, precise molecular composition of diverse crystalline inclusions was rarely investigated by analytical methods. It is possible that some of them have been assigned erroneously, and that re-examination of their true nature is warranted [24].

The physiological significance of crystalline inclusions consisting of purine derivatives also remains elusive although a number of their potential functions were suggested, including phototaxis [27], detoxification [28], bioluminescence [25], and nitrogen storage [29], [30], [31]. Insight into their physiological role is obviously limited by the lack of rapid and reliable high-throughput methods for their unambiguous identification and documentation in situ, which would not require extraction or complicated sample preparation.

Here, we demonstrate the simplicity and straightforwardness of micro-RS-based identification of guanine as a principal component of the crystal-like inclusions in the cells of the chlorophyte Desmodesmus quadricauda and the eustigmatophyte Trachydiscus minutus. We also compare our results with recent independent observations of nitrogen-rich crystalline vacuolar inclusions by analytical electron microscopy in a number of other chlorophyte species [16].