Revised: 29 April 2021

Accepted: 30 April 2021

DOI: 10.1002/jrs.6142

SPECIAL ISSUE - RESEARCH ARTICLE

Label-free detection of polysulfides and glycogen of
Cyanidium caldarium using ultra-multiplex coherent
anti-Stokes Raman scattering microspectroscopy
Yuki Oka1|

Masaki Yoshida2 |2Makoto M. Watanabe

Ayumi Minoda3| Philippe Leproux4|

1,5| Hideaki Kano

1

Department of Applied Physics, Graduate
School of Pure and Applied Sciences,
University of Tsukuba, Tsukuba, Japan

2

Abstract
Living microalga Cyanidium caldarium was visualized using ultra-multiplex

Algae Biomass and Energy System R&D
Center, University of Tsukuba, Tsukuba,Japancoherent anti-Stokes Raman scattering (CARS) microspectroscopy. The Raman
band related to the SO42 ion at 982 cm 1 was detected at the peripheral part3of the cells and showed a signal amplitude that was approximately threefold

Faculty of Life and Environmental
Sciences, University of Tsukuba, Tsukuba,
Japan

4

Institut de Recherche XLIM, Limoges,
France5Department of Chemistry, Faculty of
Science, Kyushu University, Fukuoka,
Japan
Correspondence
Hideaki Kano, Department of Chemistry,
Kyushu University, 744, Motooka, Nishiku, Fukuoka-shi, Fukuoka 819-0395,
Japan.
Email: hkano@chem.kyushu-univ.jp

larger than that in the medium, indicating accumulation of SO42 ions.
Depending on the incubation conditions, a broad and sharp Raman band at
approximately 505 cm 1 was observed at the peripheral part and inside thecells, respectively. Based on comparison of the results with model substances,these broad and sharp bands were assigned to polysulfides and glycogen,respectively.
KEYWORDS

algae, CARS, microscopy, microspectroscopy, supercontinuum

Funding information
Japan Society for the Promotion of
Science, Grant/Award Number: 18H02000

1 | INTRODUCTION
Recently, Raman spectroscopy has attracted muchattention as a single-cell analytical method for investigating microalgae without cell disruption. Thanks to themolecular specific vibrational contrast, various kinds ofintracellular metabolites can be visualized in a label-freemanner. Moudříkova et al.[1] applied confocal spontaneous Raman microscopy to the microalga Chlorella
vulgaris and revealed the intracellular distribution ofpolyphosphoric acid. Samek et al.[2] determined theeffective iodine value in lipid storage bodies of theindividual living microalgae Chlamydomonas sp. and
2572© 2021 John Wiley & Sons, Ltd.

Botryococcus sudeticus by spontaneous Raman microscopy. Heraud et al.[3] performed spontaneous Raman
microscopy of Dunaliella tertiolecta and predicted thenutrient status of individual algal cells.
Moreover, coherent Raman scattering microscopies,
such as coherent anti-Stokes Raman scattering (CARS)
microscopy and stimulated Raman scattering (SRS)
microscopy, have also been applied to investigate intracellular metabolites. In coherent Raman scattering, twolaser pulses with different colors are used: ω1 (pump) andω2 (Stokes) pulses. If the wavenumber difference(ω1 ω2) of the two incident laser pulses coincides withthe specific wavenumber (Ω) of the vibrational mode of

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Received: 19 February 2021

the sample molecule, namely, ω1 ω2 = Ω, thevibrational mode of a large number of sample molecules
is resonantly and coherently excited. The vibrational
coherence generated in this process is converted to ωCARS
or ωSRS radiation through the interaction of moleculeswith the third laser pulses (ω1 for CARS and ω2 for SRS[stimulated Raman loss]). Cavonius et al.[4] applied
CARS microscopy to the microalga Phaeodactylum
tricornutum and found that the organism uses two
different modes for normal and excessive lipid accumulation processes. Hiramatsu et al.[5] applied a Fouriertransform CARS flow cytometer to evaluate the microalga Haematococcus lacustris and measured the synthesisof astaxanthin molecules. He et al.[6] applied CARS
microscopy to the microalga Coccomyxa subellipsoidea
C169 and reported that the Raman signal of lipid droplets
appeared after N-depletion. Wakisaka et al.[7] performedlabel-free video-rate metabolite imaging of the microalga
Euglena gracilis by SRS microscopy and observed theintracellular metabolite distributions under different
culture conditions. Wang et al.[8] applied frequencymodulated SRS microscopy to Botryococcus braunii
and visualized intracellular lipid droplets and extracellular hydrocarbons. Particularly, ultra-multiplex CARS
spectroscopic imaging was shown to be a powerful
method for identifying molecular species in a label-freemanner under a microscope.[9–12] We performed ultramultiplex CARS microspectroscopic imaging[12–14] of
Aurantiochytrium mangrovei, revealing intracellularaccumulation of squalene in vacuoles.[15] In the present
study, we applied this technique to the living alga
Cyanidium caldarium to detect hitherto unknown
metabolites.
C. caldarium is a historically well-studied strain in
Cyanidiophyceae, which can survive at low pH values
from 0.5 to 4.0 and temperatures from 35 to 56 C in theirnatural environments, including acid hot springs andmoist acid soils.[16,17] C. caldarium is a red alga but lacks
the red pigment phycoerythrin. Therefore, the cells
exhibit a blue-green color. It is known as a good model
system to understand the traits of photosynthetic eukaryotes and extremophiles.[17–19] Further, its biologicalapplication for biofuel production, urban wastewater
treatment, and precious metal recycling has been
receiving attention in recent years.[20–23]
Cyanidiophyceae
comprises
Cyanidium,
Cyanidioschyzon, and Galdieria. Of the three species, only
Cyanidioschyzon has no obvious cell wall, whereas
Cyanidium and Galdieria contain a layered rigid cellwall.[24,25] The rigid cell wall gives these species ahigher tolerance to environmental stresses, including
high salinity and high osmolarity, compared with
Cyanidioschyzon.[17] However, it makes morphological2573

and biochemical analyses in these species difficult. Thus,
label-free single-cell analysis of C. caldarium will
overcome the problem and provide new insights into the
biology of Cyanidiophyceae.

2 | EXPERIMENTAL
2.1 | Setup
Figure 1 shows the experimental setup of the homebuilt
multimodal nonlinear optical microscopic system.[14]This system enables acquisition of ultra-multiplex CARS,
second harmonic generation (SHG), and third harmonic
generation (THG) signals. The main laser source was acustom-made, dual-fiber output synchronized laser
source (OPERA HP, Leukos, Limoges, France). In thelaser housing, the master laser output, with a wavelengthof 1064 nm, temporal duration of 50 ps, and repetition
rate of 1 MHz, was divided into two. One was introduced
into the large-mode area photonic crystal fiber (PCF) andused as the pump beam, ω1, which was collimated by acollimator lens (F810APC-1064, Thorlabs, Newton, NJ,
USA). The other was injected into the PCF with highnonlinearity to obtain ultra-broadband supercontinuum(SC) radiation ranging from visible to near infrared(NIR). The NIR spectral components of the SC wereselected using an optical filter (IR80, Kenko-optics,
Tokyo, Japan) and were used as the Stokes beam (ω2).
The SC was collimated by an off-axis parabolic mirror(RC04APC-P01, Thorlabs) to avoid chromatic dispersion.
The two beams were superimposed by a notch filter(NF03-532/1064E-25, Semrock, Rochester, NY, USA) andguided into a modified inverted microscope (ECLIPSE Ti
with modification, Nikon, Tokyo, Japan) in collinear
geometry. Two laser beams were tightly focused on thesample through a microscope objective (CFI Plan Apo
60 NA 1.27, water immersion, Nikon). The sample wasplaced on a piezoelectric stage (Nano-LP200, Mad City
Labs, Madison, WI, USA) for three-dimensional (xyz)
position selection. The full scanning range of the
xyz-piezo stage was 200 μm3. The sample on the piezo
stage was raster-scanned (first in the horizontal direction
and then in the vertical direction). The SHG, THG, andultra-multiplex CARS signals were collected using a second
objective lens (Plan S Fluor 40 NA 0.6, Nikon) andspectrally separated by a dichroic mirror. The CARS signaltransmitted through the dichroic mirror was detected using
a spectrometer (LS785, Princeton Instruments, Trenton,
NJ, USA) equipped with a charge-coupled device (CCD)
camera (BLAZE 100HR, Princeton Instruments). The
spectral coverage and spectral resolution of the CARS
signal were 3500 and 8 cm 1, respectively. The SHG and

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OKA ET AL.

FIGURE 1

OKA ET AL.

Experimental setup of ultra-multiplex spectroscopic CARS system with SHG and THG channels

THG signals reflected by the dichroic mirror weredetected using a spectrometer (SpectraPro300i, Princeton
Instruments) equipped with a CCD camera (PIXIS 100B,
Princeton Instruments). The exposure time at each spatial
position was 50 ms for signal detection. The image size
(pixels and microns) and step size were 101  101 pixels,
50  50 μm2, and 0.5 μm, respectively.

was sandwiched on a slide glass and cover slip, whichwere sealed with nail polish.
Na2S4 (Dojindo Molecular Technologies, Rockville,
MD, USA) and glycogen (FUJIFILM Wako, Osaka,
Japan) were used without further purification.

3 | RESULTS A ND DISCUSSION
2.2 | Sample preparation
C. caldarium NIES-2137 was obtained from the NIES
culture collection (National Institute for Environmental
Studies, Onogawa, Japan). The strain was maintained inglass test tubes containing 10 mL of M-Allen medium,[26]with the pH adjusted to 2.0 with H2SO4. The temperatureof the incubator (BioTRON LH-240SP, NK System,
Tokyo, Japan) was set to 35 C with continuous whitefluorescent light (Panasonic, Osaka, Japan, 80 μmol
m 2 s 1) without agitation. Before the measurements,
the strain in the glass tube was maintained for 16 and47 days at room temperature (20.9 C) after the last
incubation and was used as the sample. For microscopy,a 5 μL drop of M-Allen medium containing living cells

Figure 2a shows an optical image of living C. caldarium
cells, which ranged in size from 2 to 10 μm in diameter.
The green pigments correspond to chloroplasts. Figure 2b
shows a CARS intensity mapping of the same area shownin Figure 2a, which was mapped using the CARS signal
intensity at 1653 cm 1 (vertical dashed line in Figure 2c),corresponding to the apparent peak of the dispersion
lineshape. The laser power was approximately 170 and
80 mW for the pump and Stokes, respectively. The bandaround 1653 cm 1 corresponds to amide I and/or cis
C=C stretching vibrational modes. Therefore, the CARS
intensity mapping in Figure 2b corresponds to the superposition of the images due to proteins and lipids. It
should be noted that the cell debris floating in themedium, which is not observed in the optical image,10974555, 2021, 12, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jrs.6142 by University Of Tsukuba, Wiley Online Library on [12/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

2574

2575

F I G U R E 2 (a) Optical image of Cyanidium caldarium. (b) CARS intensity mapping at 1653 cm 1. (c) Spectral profile of the raw CARS
signal at the position indicated in (b). (d) Im[χ(3)] spectra calculated from (c). The sample was maintained for 47 days at room temperature(20.9 C) after nutrients in the medium were exchanged

gives an artificial image contrast in Figure 2b, especiallyat the upper and lower right areas of the images. The celldebris in the medium was tweezed by the laser beam andwas moved to the horizontal direction in the image
during the raster scanning of the sample. The inset of
Figure 2c shows the raw CARS spectra at two different
intracellular positions (blue and green crosses) in
Figure 2b. Because the raw CARS signals are distorted
mainly by the spectral profile of the Stokes pulses anddiffraction efficiency of the spectrometer, we performed
spectral correction of this signal by dividing the raw
CARS spectra by the spectral profile of the medium,
which is considered as non-resonant background (NRB).
The resulting intensity-corrected CARS spectra areshown in Figure 2c. The spectral profiles are dispersive
because the vibrationally resonant signal and NRB
interfere with each other. We next retrieved the purevibrationally resonant spectra, which corresponded to theimaginary part of the χ(3) (Im[χ(3)]) spectra, from thespectra shown in Figure 2c using the maximum entropy
method.[27] The Im[χ(3)] spectra with backgroundsubtraction are shown in Figure 2d.
As indicated in Figure 2d, the Im[χ(3)] spectra
showed characteristic Raman bands at 1664, 1457, 1339,
1267, 1236, 1131, 1086, 1037, 1008, and 982 cm 1,
corresponding to amide I and/or cis C=C stretching, CH3
degenerate deformation, CH deformation, =C–H bending, amide III, skeletal C–C (trans) stretching, skeletal
C–C (gauche) stretching, in-plane phenyl ring deformation, phenyl ring breathing, and SO42 symmetricstretching vibrational modes, respectively.[10,12]
By analyzing the spectral profile at each cell position,
we reconstructed the CARS images at various Raman
bands. Figure 3 summarizes the results of ultra-multiplex

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OKA ET AL.

OKA ET AL.

F I G U R E 3 (a) Optical image (same image as in Figure 2a); CARS images of C. caldarium around (b) 1664, (c) 1610, (d) 1457, (e) 1267,(f) 1037, (g) 1008, (h) 982, (i) 944, and (j) 860 cm 1, (k) SHG, (l) THG. These images are averaged over 2 μm2 (see the text). The sample wasmaintained for 47 days at room temperature (20.9 C) after nutrients in the medium were exchanged

CARS imaging. Because the signal-to-noise ratio was nothigh in the fingerprint region, the Raman band at eachposition was first averaged over 2 μm2 and then fitted by
the Gaussian function. The fitted results around 1660 and1000 cm 1, where several Raman bands are congested in
the fingerprint region, are shown in Figure S1. Finally,
the fitted result was mapped out. The CARS images
without spatial averaging are also indicated in Figure S2.

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2576

Figure 3b–j shows the CARS images around 1664 (b),1610 (c), 1457 (d), 1267 (e), 1037 (f), 1008 (g), 982 (h),944 (i), and 860 (j) cm 1. The CARS images in Figure 3
were classified into two main categories. First, thoseexhibiting image contrast similar to that of the opticalimage are shown in Figures 3b,d,g. Second, specific cellswith high image contrast are shown in Figure 3c,e,f,h,i,j.
Among them, the CARS image at 982 cm 1 (Figure 3h)
showed high image contrast at the peripheral parts ofthe cells. The CARS signal amplitude at 982 cm 1 at
the peripheral parts of the cells was approximately
threefold larger than that outside of the cells (Figure S3).
As the band at 982 cm 1 was assigned to the SO42 ion,
the image reveals the accumulation of SO42 ions at the
peripheral parts of the cells.
Figure 3k,l shows the SHG and THG images. As
discussed later, the SHG image is similar to the CARS
images at the Raman shifts of 944 (Figure 3i) and
860 cm 1 (Figure 3j). On the other hand, the THG image
is similar to the CARS images at the Raman shifts of 1664
(Figure 3b) and 1457 cm 1 (Figure 3d) visualizing
intracellular lipids.[10]During spectroscopic analysis, we found that two
Raman bands with different bandwidths overlapped at

2577

approximately 505 cm 1. Figure 4a shows the spectral
profiles at two intracellular positions, A and B. The Im[χ(3)] spectrum at Position A (red) showed a broad
spectral profile. In contrast, the broad and sharp bands
were superposed at Position B (green). To decompose thetwo spectral components with broad and sharp bandwidths, we fitted the spectral profile around 505 cm 1 bydetermining the sum of the broad and sharp Gaussianfunctions, whose full width at half maxima were about
60 and 19 cm 1, respectively. The broad and sharp
Gaussian functions are indicated in Figure 4a,d, whereas
Figure 4b,c shows the decomposed CARS image of thebroad and sharp spectral components, respectively.
The CARS image of the broad band clearly gives a highimage contrast at the peripheral parts of the cells.
We next considered the molecular origin of the broadband. A Raman band around 505 cm 1 is typicallyobserved in biomolecules containing disulfide bonds(S–S). According to a previous study,[25] the acid- andheat-resistant C. caldarium has cell walls that are unusually rich in protein (50%–55%). Therefore, one candidate
is proteins containing amino acids, such as cysteine(Cys), which can form interchain disulfide bonds(Cys–Cys). However, the observed spectral profile (A in

F I G U R E 4 (a) Spatially averaged Im[χ(3)] spectra (solids) at Areas A and B and the fitted result (dotted). The sum of the broad andsharp Gaussian functions was used to fit the spectral profile around 505 cm 1. CARS images of the (b) broad (FWHM: 60 cm 1) and(c) sharp (FWHM: 19 cm 1) Raman bands. These images are averaged over 2 μm2. (d) Spatially averaged Im[χ(3)] spectrum (solid) at Area C
and the fitted result (dotted). CARS images of the (e) broad and (f) sharp Raman bands. These images are averaged over 2 μm2.The results
shown in (a–c) and (d–f) were obtained using live algae, which were maintained for 47 and 16 days at room temperature (20.9 C) after
nutrients in the medium were exchanged

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OKA ET AL.

Figure 4a) shows a prominent band only around505 cm 1 without the typical band generated byproteins.[28,29] Therefore, the band around 505 cm 1
could not be assigned to a disulfide bond in Cys–Cys.
Another candidate is polysulfides. A previous study
showed that the red alga Chondria californica produces
cyclic polysulfides.[30] Many studies have focused on the
Raman band due to polysulfides from the perspective ofbattery fabrication. Solid-phase polysulfides showed a
Raman band similar to that observed. Janz et al.[31]reported a band at 488 cm 1 for Na2S5. Steudel andChivers[32] reported bands at 505, 494, 450, 424,
and 410 cm 1 for solid-state Cs2S6. All of these bands
were assigned as S–S stretching vibrational modes.
Chivers et al.[33] reported bands at 503, 453, 419, and395 cm 1 for S72 ion in [PPN]2S72EtOH, all of whichwere also assigned as S–S stretching vibrational modes.
Nims et al.[34] reported a band at 473 cm 1, with ashoulder feature at 467 cm 1, for solid S8 allotropes,
which were assigned as S–S bond stretching of the S8 ring
structure. Wu et al.[35] reported bands at 518 cm 1 forS4 and 534 cm 1 for S3. Janz et al.[31] reported bands at

OKA ET AL.

458 and 476 cm 1 in BaS3, which were assigned to S–Sstretching at 25 C. Khan et al.[36] reported a band at
450 cm 1 for S42 or S52 and at 495 cm 1 for S52 in apolysulfide solution with an S:Na ratio of 1.8 in
1.8 mol dm 3 aqueous Na2S solution. Hagen et al.[37]reported DFT calculations of S72 and S82 , which
produce Raman bands at 486 and 487 cm 1, respectively.
We then measured the polysulfides to compare theintracellular Raman band around 505 cm 1 with that ofa model polysulfide-containing substance. Figure 5cshows the Im[χ(3)] spectrum of Na2S4, which was
obtained with the same setup used to measure living
cells. The main band in the spectrum was observed
around 505 cm 1, which coincides well with the Raman
band observed in C. caldarium. Therefore, the broad
Raman band around 505 cm 1 is likely assignable to S–Sstretching vibrational modes of polysulfides.
A sharp Raman band was also observed around505 cm 1. Based on analysis of the spectroscopic images,
the sharp band was accompanied by Raman bands at
865 and 946 cm 1 (Figure 5b). These Raman bands are
known as markers of polysaccharides, such as glycogen

F I G U R E 5 (a) Im[χ(3)] spectrum at the intracellular positions indicated as A in Figure 4a. (b) Im[χ(3)] spectrum at the positions
indicated as C in Figure 4b. Im[χ(3)] spectra of Na2S4 (c) and glycogen (d)10974555, 2021, 12, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jrs.6142 by University Of Tsukuba, Wiley Online Library on [12/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

2578

and starch. According to the literature, C. caldarium
accumulates glycogen in the cytosol.[38,39] This property
is unique compared with green algae and terrestrial
plants, which store starch in their chloroplasts. We compared the spectral profile showing a sharp band around505 cm 1 (Figure 5b) with a model substance ofglycogen. As the main spectroscopic features werereproduced, we assigned the molecular species observedin C. caldarium to glycogen. Interestingly, the CARS
images due to glycogen (Figure 3i,j) gave similar image
contrast to the SHG image (Figure 3k), indicating that
accumulated glycogen inside cells is SHG active.

4 | C ON C L U S I ON
In conclusion, we successfully visualized intracellularpolysulfides in living C. caldarium cells, which showed aspectroscopic signature around 505 cm 1 with a broad
bandwidth of 60 cm 1. Depending on the incubation
conditions, intracellular glycogen was also visualized as a
sharp Raman band around 505 cm 1 with a sharp bandwidth of 19 cm 1. Based on our spectroscopic analysis,
polysulfides and glycogen were spectrally separated toobtain each CARS image, demonstrating that polysulfides
and glycogen were exclusively localized. Our approachprovides a foundation for developing a new label-free
rapid cell screening method to identify unknown
metabolites.
ACK NO WLE DGE MEN TS
The authors gratefully acknowledge the assistance ofJ. Ukon of Ukon Craft Science, Ltd., for establishing a
fruitful collaboration between Japanese and Frenchlaboratories. This study was financially supported by
Japan Society for the Promotion of Science KAKENHI
Grant Number 18H02000 (Grant-in-Aid for Scientific
Research [B]). This work benefited from French government support managed by the National Research Agency
under the Investments for the Future program with thereference ANR-10-LABX-0074 Sigma-LIM.
ORCID
Yuki Oka https://orcid.org/0000-0002-2522-3505
Philippe Leproux https://orcid.org/0000-0002-3854413X
Makoto M. Watanabe https://orcid.org/0000-00033267-7270
Hideaki Kano https://orcid.org/0000-0003-3682-7627
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SU PP O R TI N G I N F O RMA TI O N
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How to cite this article: Y. Oka, M. Yoshida,
A. Minoda, P. Leproux, M. M. Watanabe, H. Kano,
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