Raman and fluorescence microscopy sensing energy-transducing and energy-storing structures in microalgae

Moudříková, Šárka; Mojzeš, Peter; Zachleder, Vilém; Pfaff, Christian; Behrendt, Dominik; Nedbal, Ladislav

doi:10.1016/j.algal.2016.03.016

Cited by 37 publications

(36 citation statements)

References 49 publications

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“…A; Moudříková et al. , 2017a). The chemical map of polyP in CWM cells is a negative copy of the Raman image of chloroplasts visualized by their characteristic Raman spectrum consisting mainly of bands attributable to glycolipids and proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A; Moudříková et al. , 2017a). The simplest explanation for the presence of polyP grains in Arctic Cylindrocystis may stem from their role as an effective long‐term reservoir of phosphorus.…”

Section: Discussionmentioning

confidence: 99%

“…To remove the strong autofluorescence of chlorophyll obscuring the Raman spectra of photosynthetic microorganisms, wide area, low‐power photobleaching of the entire cell with a defocused 532‐nm laser beam was applied prior to mapping according to Moudříková et al. (). Raman chemical maps were constructed by multivariate decomposition of the baseline‐corrected spectra into the spectra of pure chemical components using WITec Project Plus 5.1 software.…”

Section: Methodsmentioning

confidence: 99%

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The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

Barcytė

Pilátová

Mojzeš

et al. 2019

Journal of Phycology

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The green algal genus Cylindrocystis is widespread in various types of environments, including extreme habitats. However, very little is known about its diversity, especially in polar regions. In the present study, we isolated seven new Cylindrocystis-like strains from terrestrial and freshwater habitats in Svalbard (High Arctic). We aimed to compare the new isolates on a molecular (rbcL and 18S rDNA), morphological (light and confocal laser scanning microscopy), and cytological (Raman microscopy) basis. Our results demonstrated that the Arctic Cylindrocystis were not of a monophyletic origin and that the studied strains clustered within two clades (tentatively named the soil and freshwater/glacier clades) and four separate lineages. Morphological data (cell size, shape, and chloroplast morphology) supported the presence of several distinct taxa among the new isolates. Moreover, the results showed that the Arctic Cylindrocystis strains were closely related to strains originating from the temperate zone, indicating high ecological versatility and successful long-distance dispersal of the genus. Large amounts of inorganic polyphosphate (polyP) grains were detected within the chloroplasts of the cultured Arctic Cylindrocystis strains, suggesting effective luxury uptake of phosphorus. Additionally, various intracellular structures were identified using Raman microscopy and cytochemical and fluorescent staining. This study represents the first attempt to combine molecular, morphological, ecological, and biogeographical data for Arctic Cylindrocystis. Our novel cytological observations partially explain the success of Cylindrocystis-like microalgae in polar regions.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

Barcytė

Pilátová

Mojzeš

et al. 2019

Journal of Phycology

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“…The interference from chlorophylls and carotenoids in algae can be abolished by photobleaching with a defocused laser beam before mapping. 85 The polyP counterion influences the Raman spectrum. 89 Fluorescence in situ hybridization (FISH) uses fluorescent probes that specifically bind to their complementary nucleic Analytical Chemistry pubs.acs.org/ac Feature acid sequences.…”

Section: ■ Microscopymentioning

confidence: 99%

Methods for the Analysis of Polyphosphate in the Life Sciences

2020

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Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function. See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. b Refers to absorption and fluorescence spectroscopy. Analytical Chemistry pubs.acs.org/ac Feature https://dx.

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“…However, a number of key questions remain unanswered. (i) Most past studies have selected one or just a few Raman peaks (typically derived from Raman spectra of reference chemicals) for the quantification of target compounds, yet did not evaluate the reliability of such choices [ 19 – 21 ]. For example, the relative abundance of lipids, paramylon and chlorophyll in Euglena gracilis was estimated via 2850, 2910 and 2937 cm −1 [ 19 ], while that of lipids and astaxanthin in Haematococcus pluvialis was estimated via 1448 and 1520 cm −1 [ 20 ]; however, whether and to what degree these peaks can specifically quantify the target compounds were actually not assessed.…”

Section: Introductionmentioning

confidence: 99%

Label-free, simultaneous quantification of starch, protein and triacylglycerol in single microalgal cells

Zhang

Huang

et al. 2017

Biotechnol Biofuels

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BackgroundCurrent approaches for quantification of major energy-storage forms in microalgae, including starch, protein and lipids, generally require cell cultivation to collect biomass followed by tedious and time-consuming analytical procedures. Thus, label-free, non-destructive and simultaneous quantification of such macromolecules at single-cell resolution is highly desirable in microalgal feedstock development and bioprocess control.ResultsHere, we established a method based on single-cell Raman spectra (SCRS) that simultaneously quantifies the contents of starch, protein, triacylglycerol (TAG) and lipid unsaturation degree in individual Chlamydomonas reinhardtii cells. Measurement accuracy for the contents based on full SCRS spectrum each reached 96.86–99.24%, all significantly higher than single peak-based models. However, accuracy and reliability of measurement are dependent on the number of cells sampled, thus a formal mathematical framework was proposed and validated to rationally define “minimal sampling depth” for a given state of cellular population. Furthermore, a barcode consisting of 13 marker Raman peaks was proposed to characterize the temporal dynamics of these energy-storage products, which revealed that the average contents of starch and TAG increased, while their heterogeneity indices decreased, with those of protein being exactly the opposite. Finally, our method is widely applicable, as measurements among cells from liquid suspension culture, wet paste and frozen dried powder all exhibited excellent consistency.ConclusionsWhen sampled at proper depth, SCRS can serve as a quantitative and generally applicable tool for characterization and screening of strains and bioprocesses based on the profile of energy-storage macromolecules and their among-cell heterogeneity.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0967-x) contains supplementary material, which is available to authorized users.

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Raman and fluorescence microscopy sensing energy-transducing and energy-storing structures in microalgae

Cited by 37 publications

References 49 publications

The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

Methods for the Analysis of Polyphosphate in the Life Sciences

Label-free, simultaneous quantification of starch, protein and triacylglycerol in single microalgal cells

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