Microalgal cytometric analysis in the presence of endogenous autofluorescent pigments

doi:10.1016/j.algal.2016.05.013

Algal Research

Volume 19, November 2016, Pages 370-380

https://doi.org/10.1016/j.algal.2016.05.013 Get rights and content

Highlights

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We applied multilaser and spectral flow cytometry for optimization of microalgal analysis in the presence of autofluorescence.
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Separating fluorescent dyes between different excitation lasers for microalgal staining was an effective approach.
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SYTOX Blue viability dye demonstrated minimal spectral overlap and good resolution.
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Spectral flow cytometry with virtual filtering is an advantageous approach for heterogeneous autofluorescence.

Abstract

Flow cytometry (FCM) is a well-established tool in the field of aquatic phytoplankton ecology and microalgal biotechnology, which allows for rapid assessment of the viability and physiological state of individual cells in algal populations. However, the autofluorescent spectra of different types of chlorophyll and other algal pigments may overlap with fluorescent dyes and affect the resolution of algae clusters, sensitivity, and signal-to-noise ratio. Dying algal cells continue to exhibit a strong autofluorescent signal, which may affect the evaluation of algal viability.

Herein, we tested two different approaches to measure algal fluorescence in the presence of a strong autofluorescent signal: 1) by separating dyes between different excitation lasers in order to reach minimal spectral overlap with the autofluorescent signal using flow and imaging cytometry and 2) through full spectrum analysis, virtual filtering and spectral unmixing of dye combinations and algal pigments' autofluorescence via spectral flow cytometry. For this purpose, we used viability dyes from the SYTOX family and lipophilic dyes. Among the dyes tested, the SYTOX Blue (SB) dye had minimal overlap with chlorophyll fluorescence and can be combined with autofluorescence assessment and lipophilic dyes (validated with Emiliania huxleyi algal monocultures). Imaging cytometry provided a detailed characterization of algal subpopulations stained with a combination of fluorescent dyes. A spectral flow cytometer allowed us to analyze environmental phytoplankton samples stained with fluorescent dyes in the presence of strong and heterogeneous autofluorescence from intrinsic algal pigments. We concluded that the multi-color staining of algal samples can be achieved in the presence of strong and diverse algal autofluorescence using dyes with minimal spectral overlap, a multi-laser approach (flow and imaging cytometry) and/or virtual filter and spectral flow cytometry instrumentation. This can open a new page for analytical and cell sorting algal applications.

Introduction

Though widely used in algal biotechnology and research, the full potential of FCM for microalgal characteristics is not fully exploited. The main problems are the wide range of cell sizes and high level of autofluorescence; which makes the selection of appropriate dyes originally developed for the characterization of mammalian cells difficult [1]. Microalgae exhibit high autofluorescence due to the presence of multiple photosynthetic pigments such as chlorophyll a, b, c, and d, phycobiliproteins (phycocyanins, phycoerythrin (PE)), carotenoids, and others [2]. The heterogeneous evolutionary origin of microalgae, together with their capabilities of supporting efficient photosynthesis at different depths in water columns, allows for higher chemical diversity of phytoplankton photosynthetic compounds. The autofluorescence intensity of different pigments and their emission characteristics are subjected to variations correlated to changes in algal viability, changes in metabolic activity and physiological status [3], and can lead to image saturation (in microscopy and imaging cytometry).

The autofluorescent pigment emission overlaps with the emission wavelengths of many nucleic and metabolic dyes, and as a result, may lead to a decrease in sensitivity, low signal-to-noise ratio [4], and misidentification of positive subpopulations. Thus, classic fluorescent dyes used for microalgal staining such as Nile Red (neutral lipid dye; [9-(diethyl amino) benzo[a]phenoxazin-5(5H)-one]) [5], [6] and propidium iodide (DNA-stain commonly used for identifying dead cells in a population) have a maximal emission near 600 nm, which significantly overlaps with the intrinsic autofluorescence of algal pigments - in particular, chlorophyll. In order to separate the fluorescent dye signal from strong red chlorophyll fluorescence, many researchers have employed viability and metabolic fluorescent dyes emitting in the green part of spectra (metabolic dyes: fluorescein diacetate (FDA), calcein AM, BODIPY ^505/515; viability dyes: SYTOX Green (SG) and/or SYBR Green) [7], [8], [9], [10], [11]. The BODIPY ^505/515 [4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 505/515], a recently introduced dye for rapid quantification of lipids accumulation in the algae [11], [12], [13], [14], also emits in the green channel. In the absence of other dyes, some authors retreated to the measurement of viability and metabolic activities of algal cultures sequentially, using the same green channel (SG and FDA) [15]. However, the fluorescent signal of these dyes can be affected by intrinsic green autofluorescence - which is also a common feature of green algae, cyanobacteria, dinoflagellates, and diatoms [16] - and by the bleeding of autofluorescence from neighboring channels.

Alternatively, different groups have exploited the measurement of the intrinsic fluorescence of chlorophyll [17], [16], [18] and other fluorescent pigments (green fluorescence) [19], [20] for a number of applications such as viability evaluation, characterization of phytoplankton diversity, screening of microalgal strains for biotechnological processes, and evaluation of environmental toxicity [17], [21]. However, chlorophyll continues to fluoresce in some non-viable cells, which may lead to inaccurate viability quantitation results using autofluorescence as the sole parameter of viability.

In this report, we describe two different approaches to the quantitation of fluorescent dyes in the presence of algal autofluorescence: 1) multi-laser analysis, where a number of different fluorescent dyes are separated between lasers in order to reach minimal overlap with the autofluorescent signal and 2) the virtual optical filter approach which allows for а better separation of the autofluorescent signal of pigments via spectral flow cytometry. Nucleic dyes from the SYTOX family (SYTOX Orange (SO), SG, SB) and new lipophilic dyes (BODIPY ^493/503, DiA, Dil and DiO) emitting in different channels were used in order to characterize monocultured (E. huxleyi) and environmental algal populations. We hypothesize that these suggested approaches will improve the detection of fluorescent dyes in the presence of a strong and heterogeneous autofluorescent algal signal and will also allow for the simultaneous detection of microalgae samples stained with a combination of fluorescent dyes.

Section snippets

Cell culture

E. huxleyi strains CCMP3266 (calcified) and CCMP2090 (non-calcified) were obtained from the National Center for Marine Algae and Microbiota (Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA). Algae were cultivated in L1-Si medium which is based on L1 medium [22] without the addition of Na₂SiO₃. Cultivation was conducted in a Percival incubator at 18 °C with a light intensity of 150 μmoles/m²/s at a 12/12 L/D cycle. Algae were subcultured once in three weeks and grown in the fresh

Detection of autofluorescence of algal samples

Flow cytometry and imaging flow cytometry analysis revealed a strong autofluorescence of algal E. huxleyi monocultures - in particular, the autofluorescent signal in the part of spectra corresponding to chlorophyll. As a control for viability staining, we used boiled (1 h × 100 °C) E. huxleyi monocultures. Boiling led to the disruption of the algal cellular wall and cell death, as well as the fading of the initial autofluorescent signal, as depicted in Fig. 1 (representing fluorescent intensity

Discussion

In this paper, we addressed the problems of microalgal multi-color fluorescent staining in the presence of strong, heterogeneous autofluorescence of intrinsic pigments. The intrinsic autofluorescence of microalgal cells can complicate cytometric analysis for microalgal viability and metabolic activity. The absolute signal intensities of algae intrinsic autofluorescence can be impacted by the biological and physiological conditions of cells [3] as well as the power of the laser, amplitude gain

Conclusions

The reliable detection of multi-parametric fluorescent staining of microalgae can be challenging due to the significant autofluorescence of algal pigments. The strong autofluorescence from algal pigments can overlap with the fluorescent signals from dyes and may lead to significant risks for data analysis. The method used in the present study enabled the quantitation of fluorescence in the presence of a strong signal arising from algal autofluorescent photosynthetic pigments. The nucleic and

Acknowledgements

Funding was provided in part by MES of Republic of Kazakhstan PI NURIS budget program 055 project # 100/14 to N.S.B., and by RFBR # 13-04-40189-H to I.A.V. We would like to thank CRDF Global for supporting V.D. with travel grant funding # GISX-14-60324-0. We are very grateful to Steve Conway and Jeff Clapper from SONY Inc. and Brian Hall, Rich DeMarco and Sabrina Hawthorne from Amnis (EMD-Millipore-Merck) for help with acquisition and access to their instrumentation, and providing scheme of

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View full text

Microalgal cytometric analysis in the presence of endogenous autofluorescent pigments

Highlights

Abstract

Introduction

Section snippets

Cell culture

Detection of autofluorescence of algal samples

Discussion

Conclusions

Acknowledgements

FEMS Microbiol. Rev.

Biotechnol. Annu. Rev.

J. Microbiol. Methods

Bioresour. Technol.

J. Microbiol. Methods

Trends Biotechnol.

Biotechnol. Adv.

Phycobilisomes: light harvesting pigment complexes

Bioscience

Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence

Anal. Biochem.

Isolation of high-lipid content strains of the marine microalga Tetraselmis suecica for biodiesel production by flow cytometry and single cell sorting

J. Appl. Phycol.

Enumeration and cell cycle analysis of natural populations of marine phytoplankton by flow cytometry using the nucleic acid stain SYBR Green I

Appl. Environ. Microbiol.

Cellular DNA content of marine phytoplankton using two new fluorochromes: taxonomic and ecological implications

J. Phycol.

Flow cytometric analysis of phytoplankton viability following viral infection

Aquat. Microb. Ecol.

Development of an improved rapid enzyme inhibition bioassay with marine and freshwater microalgae using flow cytometry

Arch. Environ. Contam. Toxicol.

In situ analysis of heterogeneity in the lipid content of single green microalgae in alginate hydrogel microcapsules

Anal. Chem.

BODIPY vital staining as a tool for flow cytometric monitoring of intracellular lipid accumulation in Nannochloropsis gaditana

J. Appl. Phycol.