Progress in Earth andPlanetary Science

Kato et al.
Progress in Earth and Planetary Science(2023) 10:13
https://doi.org/10.1186/s40645-023-00543-5

Open Access

METHODOLOGY

A simple method for taxon‑specificpurification of diatom frustules from ocean
sediments using a cell sorter
Yuji Kato1,2* , Yuki Morono3 , Akira Ijiri3,4 , Takeshi Terada5 and Minoru Ikehara2   

Abstract
It is necessary to purify diatom frustules by taxon to perform accurate geochemical analyses of diatom fossils preserved in sediments. However, the small size of diatoms has hitherto prevented taxon-specific purification; therefore,
previous geochemical analyses of diatom frustules have been performed with mixtures of various taxa. In this study,
we developed a taxon-selective collection method of diatom fossils that uses a cell sorter. The experimental material
comprised six samples from a sediment core of Hole U1538A in the Scotia Sea, Atlantic sector of the Southern Ocean
drilled during the International Ocean Discovery Program Expedition 382. Following conventional pretreatments(removal of organic matter and carbonates as well as clay minerals), we conducted cell sorter experiments focusing
on the optical and fluorescence characteristics of the diatom fossils. We succeeded in selectively isolating with highpurity five diatom taxa representing the Southern Ocean diatom flora: (1) discoid diatoms (mainly Thalassiosira) with amoderate degree of fluorescence; (2) Fragilariopsis (mainly Fragilariopsis kerguelensis), which exhibits the highest fluorescence values; (3) Rhizosolenia; (4) Eucampia antarctica; and (5) needle-shaped diatoms (Thalassiothrix). This taxonspecific diatom purification method will enable more accurate geochemical analyses, such as the oxygen isotope
ratio (δ18O) of diatom frustules, which is likely to lead to significant advances in paleoceanography, especially at highlatitudes or in upwelling zones where diatoms are abundant. This method will also be useful in paleolimnology, ocean
biology, and phycology.
Keywords Diatom, Cell sorter, Microfossil, Sediment, Southern Ocean, International Ocean Discovery Program*Correspondence:
Yuji Kato
kato.yuji.ge@u.tsukuba.ac.jp
1
Present Address: Faculty of Life and Environmental Sciences, Universityof Tsukuba, Tsukuba, Ibaraki 305‑8572, Japan
2
Center for Advanced Marine Core Research, Kochi University, Nankoku,
Kochi 783‑8502, Japan
3
Kochi Institute for Core Sample Research, Institutefor Extra‑Cutting‑Edge Science and Technology Avant‑Garde Research(X‑Star), Japan Agency for Marine-Earth Science and Technology(JAMSTEC), Nankoku, Kochi 783‑8502, Japan
4
Graduate School of Maritime Sciences, Kobe University, Kobe, Hyogo
658‑0022, Japan
5
Marine Works Japan, Ltd., Yokosuka, Kanagawa 237‑0063, Japan

1 Introduction
Fossil diatoms are important paleoenvironmental indicators, and many paleoceanographic studies have been carried out using their assemblage compositions (e.g., Smoland Stoermer 2010). In addition to assemblage-based
studies, a considerable number of geochemical studies using diatom frustules (e.g., of oxygen isotope composition [δ18O]) have been conducted over the last few
decades. In general, calcareous microfossils are rare oralmost barren in high-latitude oceans (Dutkiewicz et al.
2016); therefore, δ18O data of diatom frustules have been
regarded as an important alternative paleoenvironmental
indicator to δ18O of foraminiferal shells (Labeyrie 1974;
Juillet-Leclerc and Labeyrie 1987; Shemesh et al. 1992,
1995, 2002; Hodell et al. 2001; Swann et al. 2006; Swann© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

and Leng 2009). Other examples of geochemical studies
on diatom frustules include the measurement of the silicon isotope composition (δ30Si) of diatom frustules as aproxy for local silicic acid utilization (De la Rocha et al.
1998; De la Rocha 2006; Reynolds et al. 2008; Maier et al.
2013). In addition, the carbon and nitrogen isotopic compositions (δ13C and δ15N) of organic matter occluded
within diatom frustules are regarded as proxies for the
carbon and nitrogen cycles (Singer and Shemesh 1995;
Sigman et al. 1999; Crosta and Shemesh 2002; Studer
et al. 2015). However, in most previous studies, geochemical measurement of diatom frustules has been carriedout on samples of mixed diatom taxa because the conventional methods (e.g., chemical treatment with hydrogen peroxide [­ H2O2] and hydrochloric acid [HCl]) do notallow isolation of diatom frustules by taxon.
Each diatom taxon has different ecological characteristics (e.g., blooming season and habitat depth); thus, it isnecessary to collect diatom frustules by taxon for accurate geochemical analysis of diatoms. In other words,
restricting the number of species is essential for obtaining more accurate paleoenvironmental information
and avoiding mixed information from various seasons/depths. However, because of the small size of diatom
valves (several tens of micrometers), isolation of any
taxon by morphological features using conventional
methods has been very challenging (cf. hand-picking
under a stereomicroscope, which is a widely accepted
method for foraminifer-based studies).
In recent years, flow cytometry, in the form of a cell
sorter, has been applied to geological samples to isolate
target particles, such as micromanganese nodules (Uramoto et al. 2019), diatom fossils (Tennant et al. 2013a;
Ijiri et al. 2021), and fossil pollen (Tennant et al. 2013b;
Zimmerman et al. 2018; Tunno et al. 2021; Kasai et al.
2021; Yamada et al. 2021). A cell sorter is an instrumentthat can separate microparticles by their optical characteristics, reflecting the material’s size, structure, or chemical composition (fluorescence). Cell sorters were initiallyused in the life sciences to collect living cells selectively.
By focusing on optical scattering, which reflects differences in the size and structure of diatoms, Ijiri et al.
(2021) established a method to purify discoid diatoms
using a cell sorter. However, purification of other diatom
taxa, such as pennate diatoms, has not yet been achieved,
and thus far, it has not been possible to isolate diatom
frustules by taxon selectively.
By fully utilizing the capability of a cell sorter to distinguish particles by their optical characteristics (i.e.,
focusing on both scattering and fluorescence signals fromparticles), we have established a purification method toselectively obtain various diatom taxa with high purity(ca. 85%–100%). This method is based on discovering

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that diatom frustules of different taxa exhibit distinct
optical characteristics (Sect. 3.1). Five diatom taxa weresuccessfully purified in this study: Thalassiosira spp.,
Fragilariopsis spp., Eucampia antarctica, Rhizosolenia
spp., and Thalassiothrix spp.

2 Methods
2.1 Sediment samples

For the development of the purification method, weused six sediment samples from a sediment core at HoleU1538A, retrieved from the Scotia Sea, Atlantic sector of the Southern Ocean (57° 26.5248′ S, 43° 21.4691′
W; water depth, 3130.58 m; core length, 676.0 m) during the International Ocean Discovery Program (IODP)
Expedition 382 (Fig. 1). The studied samples range from
382-U1538A-1-1, 24–25 cm (0.24 m below seafloor[mbsf ]) to 382-U1538A-5-6, 24–25 cm (43.42 mbsf ).
We selected these samples to confirm that the method
developed in this study could be applied to various typesof sediment. The approximate age of the sediment core
was estimated by paleomagnetic/biostratigraphic analysis
and from physical properties such as magnetic susceptibility (Weber et al. 2021). The upper two samples (0.24
and 1.73 mbsf ) correspond to the Holocene, the middle
two (15.10 and 17.54 mbsf ) to the last glacial period, andthe lower two (42.54 and 43.42 mbsf ) to the last interglacial (Fig. 1). The analyzed interval of the sediment core
consists of interbedded dark greenish-gray diatom oozes(interglacial periods) with variable terrigenous components and biosilica-rich/bearing silty clays (glacial periods) (Weber et al. 2021).
2.2 Pretreatment of the sediment samples

We pretreated the samples to concentrate the diatom
frustules. We followed the pretreatment method of Ijiri
et al. (2021), with slight modifications, but omitted theelutriation process (enriching discoid diatoms, whichcorresponds to “Step 2” of Ijiri et al. 2021) to preserve
the diverse diatom taxa in the samples (Fig. 2). Because
the elutriation process is time-consuming, its omission
markedly reduces the time needed for the pretreatment.
In brief, the samples were pretreated as follows (Fig. 2):organic matter and carbonates were removed by 30%­H2O2 and 5% HCl. The ­H2O2 and HCl treatments werecarried out separately (i.e., not with a mixed solution).
The samples were sieved through 63, 45, 32, and 20 µm
stainless-steel meshes in succession (with the 63 µm sieve
being first and the 20 µm sieve being last) to obtain the
20–32 µm fraction. The use of 63 and 45 μm meshes wasintended to improve the recovery of the 20–32 μm fraction: if a sediment sample is processed through a 32 µm
sieve without pre-sieving, large particles (> 45 μm) clog
the mesh so that smaller particles (< 32 µm), which would

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

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Fig. 1 Location and core profile of the studied samples. a Map showing the location of the drilling site (IODP Expedition 382 Site U1538). APF:
Antarctic Polar Front, SAF: Subantarctic Front, STF: Subtropical Front. b Horizons of the six samples analyzed in this study (red triangles) together
with lithology and magnetic susceptibility (Weber et al. 2021)

usually pass through the sieve, also remain on the sieve.
The 20–32 µm fraction was selected so that the particles
would not jam in the nozzle of the cell sorter (diameter
100 µm). Then clay minerals were removed by heavyliquid separation using sodium polytungstate (SPT;­3Na2WO4·9WO3·H2O) solution (2.1 g/cm3) (Morley
et al. 2004). Finally, the extracted sample was sieved again
through 20 µm mesh to remove any particles that mightclog the flow path of the cell sorter.
2.3 Microscopic observation

To determine the optical characteristics of the diatom
frustules, we observed the pretreated samples undera fluorescence microscope (BX53) equipped with amercury lamp (BH2-RFL-T3) and a WIB filter unit
(U-MWIB, excitation filter: 460–495 nm, emission filter: > 510 nm) (Olympus, Tokyo, Japan). Approximately
0.2 mL of pretreated sample was picked up in a micropipette and placed on a glass slide. Microscopic observations were carried out at 200 × magnification (Fig. 3).
2.4 Flow cytometry/cell sorter

For the cell sorter experiments for taxon-specific sorting of diatom frustules, we used a Moflo XDP cell sorter(Beckman Coulter, Brea, CA, USA) located at the Kochi
Core Center, Japan. The cell sorter collects optical signals from the subject particles, including forward scatter(FSC), side scatter (SSC), and fluorescence signals (Tennant et al. 2013a; Uramoto et al. 2019). The cell sorter hasa detector in front of the laser beam (input wavelength:488 nm), which measures FSC, and several detectors to
the side, which measure SSC and fluorescence (Additional file 1: Fig. S1). FSC is a light signal detected along
the laser path that is primarily caused by light diffraction
around the particles and mainly reflects the size of theparticles. SSC is a signal detected at a 90° angle relative
to the laser beam. The source of the SSC signal is refraction or reflection from the interface between the laser
beam and the particles. The complex structure of diatoms can be reflected in the wide range of SSC signals.
In addition to FSC and SSC, the cell sorter detects fluorescence signals from the sample particles. Flow cytometric analysis and sorting with FL1 and FL4 filters todetect fluorescence signals were also carried out, because
the fluorescence characteristics of diatom frustules may
differ depending on the taxon. The FL1 detector detects
a fluorescence signal with a wavelength of 530 nm (fullwidth at half maximum 40 nm), which corresponds togreen in visible light, whereas detector FL4 detects a fluorescence signal with a wavelength longer than 740 nm(Additional file 1: Fig. S1). Note that the fluorescenceobserved by the microscope does not always match thefluorescence observed by the cell sorter. To carry out theexperiments under the same device conditions (i.e., toalign fluctuation of the laser beam), we used commercial
fluorescent beads (Flow-Check Fluorospheres 6605359,
Beckman Coulter, Brea, CA, USA) to calibrate the laserand detector settings, which included FSC, SSC, andKato et al. Progress in Earth and Planetary Science(2023) 10:13

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Fig. 2 Flow diagram showing the pretreatment process for concentration of diatom frustules

fluorescence filters. We did not perform any fluorescencecorrection, a common mathematical method to handle
spectral overlap between fluorophores.
The Moflo XDP cell sorter is a “jet-in-air”-type cell
sorter in which a water stream is ejected from a narrownozzle into the air. By diluting the sample suspension sothat only one particle passed the laser at a time, we collected optical information on each particle in the suspension. Following Ijiri et al. (2021), we used a solution
of 1% NaCl in Milli-Q water for the sheath fluid, and thepretreated sample was suspended in SPT solution (1.1 g/mL) and introduced to the cell sorter. Specific ranges ofvalues can be selected for the optical/fluorescence data,and particles within these ranges are sorted away fromthe sample stream and collected, leaving the unwanted
detritus to be routed to waste. A group of particles tobe sorted is specified by an arbitrarily drawn polygon on
the sample data, such as the FSC–SSC plot and FL1–FL4

plot (e.g., Fig. 4). The polygon is called a “region.” The cell
sorter can also sort by combining multiple regions created on multiple data plots; for example, sorting using a
region on the FSC–SSC plot, which reflects the opticalcharacteristics of the sample particles (size and internal structure), and a region on the FL1–FL4 plot, whichreflects the fluorescence characteristics of the sample
particles. This sorting yields particles that satisfy both thedesired optical and fluorescence characteristics. A combination of multiple regions is referred to as a “composite
region” in this study.
After processing through the cell sorter, the obtained
diatom assemblages were analyzed. Microslides forthe diatom analyses were prepared using a photocuring adhesive (Nichika Inc., Kyoto, Japan). All diatoms
on one microslide were identified at the species level
until 50–100 valves had been counted (Additional
file 2: Tables S1–S6). We also analyzed strewn slides ofKato et al. Progress in Earth and Planetary Science(2023) 10:13

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Fig. 3 Photomicrographs of the samples after chemical pretreatment. a, b Sample 382-U1538A-1-2, 24–25 cm. c, d Sample 382-U1538A-3-1,
24–25 cm. e, f Sample 382-U1538A-5-6, 24–25 cm. Images (a), (c), and (e) were taken with transmitted light; images (b), (d), and (f) were obtainedby fluorescence imaging

the original sediment samples (i.e., microslides of bulk
sediment samples without chemical pretreatment).
The original diatom assemblage compositions are presented in Additional file 1: Fig. S2 and Additional file 2:
Table S7. A single centric diatom was counted as one
when more than half of the valve was observed. A pennate diatom specimen was counted as one when two
apices were observed. In addition, the number of fragments of the needle-shaped diatom Thalassiothrix (i.e.,
Thalassiothrix specimen without valve apices) on the

microslides after the cell sorter experiments was alsocounted (Additional file 2: Tables S1–S6).

3 Results and discussion
3.1 Sample optical characteristics and cell sorter tuning

We confirmed that the diatom morphologic features,
including shape, size, and structure (e.g., the arrangement pattern of areolae), are different among taxa, which
created different reflection and scattering patterns in
the cell sorter analysis. More importantly, we found thatthe diatoms sometimes exhibited markedly different

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

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Fig. 4 Flow cytograms and pie charts showing the diatom assemblages obtained from each sorting region. The circled numbers indicate thesorting regions established in this study. a, b Sample 382-U1538A-1-2, 24–25 cm (Holocene). c, d Sample 382-U1538A-3-1, 24–25 cm (last glacial).
e, f Sample 382-U1538A-5-6, 24–25 cm (last interglacial). Plots of FSC height versus SSC height are shown in (a), (c), (e), and FL1 height versus FL4height are shown in (b), (d), (f). Each dot in the flow cytogram indicates a particle that passes through the laser: the bluer the color, the greater thenumber of particles

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Table 1 Summary of the characteristics and diatom assemblages of the sorting regions and composite regions
Description of particle groups Sorting regions/composite regions Obtained diatom assemblage
Moderate-FSC

R1/R9/R17

Abundant discoid diatoms (Thalassiosira)  Low-FSC

R2/R10/R18

Abundant pennate diatoms (Fragilariopsis, Thalassiothrix etc.)  High-SSC

R3/R11/R19

Abundant Rhizosolenia (interglacial samples) or Eucampia (glacial samples)  High-FSC

R4/R12/R20

Increased Eucampia (interglacial samples) / Purified Eucampia (glacialsamples)Moderate-FL1

R5/R13/R21

Abundant discoid diatoms  High-FL1

R6/R14/R22

Abundant Fragilariopsis  Low-FL1

R7/R15/R23

Abundant Thalassiothrix (interglacial samples) / Abundant Eucampia (glacialsamples)  Low–Mid FL1

R8/R16/R24

Increased Rhizosolenia

Moderate-FSC × Moderate-FL1

R1 × R5/R9 × R13/R17 × R21  Low-FSC × High-FL1  Low-FSC × Low-FL1  High-SSC × Low–Mid FL1

R2 × R6/R10 × R14/R18 × R22

R2 × R7/R10 × R15/R18 × R23

R3 × R8/R11 × R16/R19 × R24

fluorescence features (Fig. 3). Specifically, the genus
Fragilariopsis showed high fluorescence intensity (visible as bright green in Fig. 3). However, discoid diatoms
showed a considerable variation in fluorescence intensity(i.e., the majority of discoid diatoms had far weaker fluorescence intensities than Fragilariopsis, but with some
exceptions; Fig. 3). In addition, specimens of Eucampia
antarctica and needle-shaped diatoms (Thalassiothrix)
showed relatively weak fluorescence intensities (Fig. 3).
These observations suggest that the fluorescence characteristics and the morphologic/optical signal are important parameters separating these taxa.
Through many cycles of flow cytometric sorting andmicroscopic observation, we determined the opticalparameters FSC and SSC, which reflect diatom morphology, and the FL1 and FL4 filters, which reflect thefluorescence characteristics of diatom frustules. Because
the morphology of diatom frustules was too complex
to predict the flow cytometric characteristics from themicroscopic observations, we repeated the sorting andobservation cycles to build “an empirical rule”. Afterthe sorting trials, we identified the sorting regions,shown in Fig. 4 and Additional file 1: Fig. S3 (Regions
1–45; Table 1). As mentioned in Sect. 2.4, these regions
specify the group of particles to be sorted. We also conducted sorting experiments using composite regions, inwhich multiple regions were combined, to obtain particles satisfying specific optical and fluorescence characteristics. The coordinates of the designated sort regions’upper right and lower left points are listed in Additional
file 2: Table S8. These parameters vary depending on the
instruments’ optical alignments and detector settings(such as the voltage applied to the photomultiplier). Even
with the same instrument and the same sample, slight

Purified discoid diatoms, mainly Thalassiosira
Purified Fragilariopsis
Purified Thalassiothrix (interglacial samples)
Purified Rhizosolenia (Holocene samples)differences of optical alignment can produce shifts in theflow cytometer plot; therefore, we adjusted the region
settings sample-by-sample, including several rounds ofsorting (ca. 50 particles) and checking under the microscope, after which we finalized the sorting region forthat sample. Therefore, the position (parameter values)of each finalized region on the plots shown in Fig. 4 differs between samples. Regions occurring in the FSC–SSC
plot reflect the particles’ optical characteristics, and thosein the FL1–FL4 plot reflect fluorescence signals (Fig. 4).
Hereinafter, “region” is abbreviated to “R”.
3.2 General results of the sorting experiments

Through the cell sorter experiments, which included
sorting of each designated region and composite
regions, we succeeded in the purification/enrichmentof five diatom taxa that represent Southern Ocean
assemblages: discoid diatoms (mainly Thalassiosira);
Fragilariopsis (mainly Fragilariopsis kerguelensis); Rhizosolenia; Eucampia antarctica; and needle-shaped diatoms (mainly Thalassiothrix) (Figs. 5–7, Additional file 1:
Figs. S4–S6). In addition, light microscopic observation
showed that almost no “non-diatom particles” were present in the samples after sorting. To confirm the method’s
applicability to collect these diatom taxa selectively, we
sorted the diatoms in each of the six samples in the various regions (Fig. 4 and Additional file 1: Fig. S3). In thefollowing section, the diatom assemblages obtained during the sorting experiments in each region and composite
regions are described. Note that the term “relative abundance” indicates the relative frequency of each diatom
taxon in the diatom assemblage, not in the total particles.

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

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Fig. 5 Flow cytograms of sample 382-U1538A-1-2, 24–25 cm (Holocene) and pie charts/photomicrographs showing the diatom assemblages
obtained by sorting of the following composite regions: Regions 1 and 5 (discoid-diatom-rich); Regions 2 and 6 (Fragilariopsis-rich); Regions 3 and 8(Rhizosolenia-rich); and Regions 2 and 7 (Thalassiothrix-rich). The crystalline materials in the micrographs are NaCl crystals in the sheath fluid

3.3 Diatom assemblages obtained from sample‑adjusted
sorting regions

In the Holocene sample (U1538A-1-2, 24–25 cm), sortingof R1 (moderate FSC values) yielded a diatom assemblage
mainly composed of discoid diatoms (Thalassiosira). The
relative abundance of this taxon was 75.2% (Fig. 4a). In

contrast, the main components of the diatom assemblage
obtained from R2 sorting were pennate diatoms such as
Fragilariopsis kerguelensis (31.9%) and Thalassiothrix
antarctica (50.0%) (Fig. 4a). R3, from which we obtained
a Rhizosolenia-dominated assemblage (94.7%), was characterized by higher SSC values (Fig. 4a). We also definedKato et al. Progress in Earth and Planetary Science(2023) 10:13

a region (R4) that had high FSC values, which was mainlycomposed of discoid diatoms. Of the regions identified
in the FL1–FL4 plot, the diatom assemblage obtained bysorting of R5 (with a moderate degree of fluorescence)
was composed of discoid diatoms, whereas the main
component of the assemblage from R6 (with high FL1
values) was Fragilariopsis kerguelensis (Fig. 4b). We also
obtained Thalassiothrix-rich (needle-shaped diatoms)
and Rhizosolenia-rich assemblages from sorting of R7(the lowest FL1 values) and R8 (slightly higher FL1 andFL4 values), respectively (Fig. 4b). In addition, we conducted sorting experiments in composite regions. Specifically, sorting of the composite region R1 × R5 resulted
in isolation of discoid diatoms with high purity (relative
abundance 95.6%; Fig. 5). Sorting of the composite region
R2 × R6 yielded highly pure assemblages of Fragilariopsis (relative abundance 99.0%; Fig. 5). We also obtained
an assemblage dominated by needle-shaped diatoms(Thalassiothrix, relative abundance 86.9%) by sorting thecomposite region R2 × R7 (Fig. 5). In addition, we succeeded in isolating Rhizosolenia with high purity (95.2%)
by sorting the composite region R3 × R8 (Fig. 5).
In the last glacial sample (U1538A-3-1, 24–25 cm), we
designated eight regions for sorting (R9 to R16; Fig. 4c,d). The diatom assemblage from R9 (moderate FSC values) was composed of discoid diatoms (57.6%) andEucampia antarctica (31.2%) (Fig. 4c). In contrast, R10(low FSC values) assemblages contained greater relative
abundances of pennate diatoms (Fragilariopsis 23.8%,
Thalassiothrix 35.6%) (Fig. 4c). We also obtained Eucampia-dominated assemblages from R11 (higher SSC) andR12 (high FSC) (Fig. 4c). In the sorting of R13 (moderate
FL1 fluorescence), we recognized an assemblage mainlycomposed of discoid diatoms and Eucampia (Fig. 4d); in
contrast, we observed markedly higher abundances of
Fragilariopsis in the R14 (high FL1) assemblage (Fig. 4d:
57.3%). Both R15 (lowest FL1) and R16 (lower FL1)
yielded Eucampia-dominated assemblages, although
the R16 assemblage contained slightly higher proportions of Rhizosolenia (Fig. 4d). In addition, sorting of thecomposite region R9 × R13 yielded purified discoid diatoms (relative abundance 86.8%; Fig. 6). We also obtained
highly pure assemblages of Fragilariopsis by sorting thecomposite region R10 × R14 (relative abundance 97.4%;
Fig. 6).
In the last interglacial sample (U1538A-5-6, 24–25 cm),eight sorting regions were defined (R17 to R24). Sortingof R17 (moderate FSC values) yielded abundant discoid
diatoms (83.9%; Fig. 4e), whereas sorting of R18 (lowFSC values) produced an assemblage dominated by pennate diatoms (Fragilariopsis 25.0%, Thalassiothrix 63.3%)(Fig. 4e). The assemblage from R19 (high SSC values)
showed high proportions of Rhizosolenia (20.6%) andPage 9 of 15

Eucampia (40.2%) (Fig. 4e). In addition, a discoid-diatomrich assemblage was obtained from the sorting of R20(high FSC) (Fig. 4e). For the FL1–FL4 plot, the diatom
assemblages of R21 (moderate FL1) and R22 (high FL1)
showed very high abundances of discoid diatoms (90.9%)
and Fragilariopsis (92.6%), respectively (Fig. 4f ). The
assemblages from R23 (lowest FL1) and R24 (low FL1)
were composed of Thalassiothrix (needle-shaped diatoms) and Eucampia (Fig. 4f ). In addition, we obtained
purified assemblages of discoid diatoms (96.4%), Fragilariopsis (97.9%), and Thalassiothrix (90.5%) by sorting
experiments of composite regions R17 × R21, R18 × R22,
and R18 × R23, respectively ( Fig. 7).
3.4 Purification/enrichment of representative diatom taxa

In this section, the regions and composite regions
described above are grouped based on the optical characteristics of the particles (“description of particle groups”
in Table 1 ), and the results obtained for each group arecompared and described.
R1, R9, and R17 had moderate FSC values (on the orderof ­103), and discoid diatoms comprised the majority ofthis moderate-FSC group (Fig. 4; Table 1). The relative
abundance of discoid diatoms was 57.6–83.9% (Fig. 4),
markedly higher than their relative abundances in theoriginal assemblages (Additional file 1: Fig. S2).
R2, R10, and R18 had lower FSC values than the moderate-FSC group (Fig. 4; Table 1). Higher abundances ofpennate diatoms characterized this low-FSC group. The
relative abundance of Fragilariopsis was 23.8–31.9% in
the low-FSC group, but was less than 5% in the moderate-FSC group. Apart from Fragilariopsis, the low-FSC
group contained needle-shaped diatoms such as Thalassiothrix (35.6–63.3%) and discoid diatoms (4.2–31.7%)(Fig. 4).
R3, R11, and R19 had higher SSC values than the otherregions (Fig. 4; Table 1 and Additional file 2: Table S8).
The assemblages of the high-SSC group varied among thesamples. In the Holocene sample, the genus Rhizosolenia
constituted the majority of the high-SSC Group (Fig. 4a).
In the last glacial period sample, the high-SSC group wascharacterized by abundant Eucampia antarctica (Fig. 4c);in contrast, the high-SSC group for the last interglacialsample yielded diatom assemblages with ca. 20% Rhizosolenia (Fig. 4e). The higher abundance of Rhizosolenia in
the high-SSC group was consistent with the taxon having a more complex valve structure and morphology thanother taxa (e.g., the discoid diatom Thalassiosira and thepennate diatom Fragilariopsis). The difference in the relative abundance of Rhizosolenia in the high-SSC groupassemblages seems to be due to differences in the compositions of the original assemblages (i.e., Rhizosolenia wasKato et al. Progress in Earth and Planetary Science(2023) 10:13

Page 10 of 15

Fig. 6 Flow cytograms of sample 382-U1538A-3-1, 24–25 cm (last glacial) and pie charts/photomicrographs showing the diatom assemblages
obtained by sorting of the following composite regions: Regions 9 and 13 (discoid-diatom-rich); Regions 10 and 14 (Fragilariopsis-rich); and Region
12 (Eucampia-rich). Sorting of composite region R12 and R15 also achieved purification of Eucampia antarctica (Additional file 2: Table S4). The
crystalline materials in the micrographs are NaCl crystals in the sheath fluid

most abundant in Holocene samples) (Additional file 1:
Fig. S2; Additional file 2: Table S7).
R4, R12, and R20 had the highest FSC values (Fig. 4;
Table 1). Although the diatom assemblages of this highFSC group varied greatly among samples, all samples

showed an increased percentage of Eucampia antarctica
relative to the original samples (Fig. 4; Additional file 1:
Fig. S2). In the sample from the last glacial, the high-FSC
group consisted almost entirely of Eucampia antarctica(Figs. 4 and 6).

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

Page 11 of 15

Fig. 7 Flow cytograms of sample 382-U1538A-5-6, 24–25 cm (last interglacial) and pie charts/photomicrographs showing the diatom assemblages
obtained by sorting of the following composite regions: Regions 17 and 21 (discoid-diatom-rich); Regions 18 and 22 (Fragilariopsis-rich); andRegions 18 and 23 (Thalassiothrix-rich). The crystalline materials in the micrographs are NaCl crystals in the sheath fluid

R5, R13, and R21 contained particles that emit moderate fluorescence (Fig. 4b, d, f, Table 1), and this moderate-FL1 group was mainly composed of discoid diatoms(Fig. 4): specifically, the relative abundances of discoid

diatoms were ca. 70–90% in the Holocene and last interglacial samples (Fig. 4b, f ) and ca. 40% in the last glacialsample (Fig. 4d).

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

R6, R14, and R22 had the highest FL1 values among
the designated sorting regions (Fig. 4; Table 1), meaningthat the diatoms of the high-FL1 group emit high fluorescence in the wavelength region 510–550 nm (Additional
file 1: Fig. S1), which corresponds to green in visible light(Fig. 3). In all samples, the high-FL1 group was composed
mainly of Fragilariopsis kerguelensis. This result is consistent with the microscopic observations described in
Sect. 3.1 that most of the Fragilariopsis spp. specimens
showed high fluorescence (i.e., high FL1 values), whereas
the fluorescence intensity of discoid diatoms varied(Fig. 3).
Particles in R7, R15, and R23 had relatively lower FL1
values (Fig. 4b, d, f; Table 1). The diatom assemblages ofthe low-FL1 group varied among samples: needle-shaped
diatoms (Thalassiothrix) were abundant in the Holocene
and last interglacial samples (Fig. 4b, f ), whereas Eucampia antarctica dominated the R15 assemblage in the lastglacial sample (Fig. 4d). This result is consistent with themicroscopic observation that Thalassiothrix and Eucampia emit relatively weak fluorescence (Fig. 3).
Particles in R8, R16, and R24 had relatively higher fluorescence values than the low-FL1 group (Fig. 4; Additional file 2: Tables S1 and S8), and are referred to here
as the low–mid FL1 group. The diatom assemblages in
the low–mid FL1 group showed higher abundances of
Rhizosolenia than those from other regions, although themagnitude of the increase varied from sample to sample.
Specifically, the Holocene sample yielded a Rhizosoleniarich assemblage (Fig. 4b). In contrast, the last glacialsample was composed mainly of Eucampia antarctica(Fig. 4d). The last interglacial sample also showed a slightincrease in Rhizosolenia; however, needle-shaped diatoms (Thalassiothrix) were dominant (Fig. 4f ).
As mentioned above, we also conducted sorting experiments using composite regions to obtain particles exhibiting both optical and fluorescence characteristics ofinterest. Sorting of the moderate-FSC and moderateFL1 groups (i.e., sorting with composite regions R1 × R5,
R9 × R13, and R17 × R21) yielded discoid diatoms withhigh purity (relative abundance 86.8–96.4%; Figs. 5, 6, 7).
Sorting of composite regions to obtain particles thatexhibit low-FSC and high-FL1 characteristics (i.e., composite regions R2 × R6, R10 × R14, and R18 × R22)
yielded highly pure assemblages of Fragilariopsis kerguelensis, the most abundant diatom taxon in the Southern Ocean (relative abundance 97.4–99.0%; Figs. 5, 6, 7).
As described above, Fragilariopsis, discoid diatoms, andneedle-shaped diatoms were present in variable proportions in the low-FSC group assemblages (R2, R10, andPage 12 of 15

Table 2 Summary of the optical and fluorescence characteristicsof the five targeted diatom taxa
Diatom taxa

FSC

SSC

Thalassiosira(discoid diatom)

Mid (~ ­103)

Mid (~ ­102–103)

Mid (~ ­102–103)

Fragilariopsis

Low (~ ­102)

Mid (~ ­102–103)

High (~ ­103–104)

Rhizosolenia

4

Eucampia
Thalassiothrix(needle-shaped
diatom)FL1 (fluorescence)4

Low (~ ­101–102)

High (~ ­10 ) High (~ ­10 )
42

3

Low (~ ­101–102)

2

2

3

Low (~ ­101–102)

High (~ ­10 ) Mid (~ ­10 –10 )Low (~ ­10 )

Mid (~ ­10 –10 )R18; Fig. 4a, c, e). By utilizing the fluorescence characteristics, we selectively sorted Fragilariopsis kerguelensis
with high purity (Figs. 5, 6, 7) while removing discoid andneedle-shaped diatoms that emit relatively weak fluorescence (Fig. 3).
For the Holocene and last interglacial samples, we
obtained highly pure assemblages of needle-shaped diatoms (Thalassiothrix, relative abundance ca. 90%) by sorting the composite regions to obtain particles with both
low FSC and low FL1 (i.e., composite regions R2 × R7 andR18 × R23) (Figs. 5 and 7). Thus, application of fluorescence characteristics allowed us to remove other pennate
and discoid diatoms from the low-FSC group assemblages (R2 and R18) and purify needle-shaped diatoms.
In addition, for the Holocene sample, we succeeded inisolating Rhizosolenia with high purity (95.2%) by sorting particles that satisfy the characteristics of both highSSC and low–mid-FL1 group with the composite region
R3 × R8 (Fig. 5).
In summary, we succeeded in purifying five diatom
taxa that are major components of the Southern Ocean
diatom flora and confirmed the applicability of this new
purification protocol through cell sorter experiments on
six samples (Figs. 5, 6, 7 and Additional file 1: Figs. S3–
S5, Tables 1 and 2 and Additional file 2: Tables S1–S6).
Specifically: (1) discoid diatoms were purified using composite regions to sort particles with both moderate FSC
and moderate FL1 values; (2) purification of Fragilariopsis (mainly Fragilariopsis kerguelensis) was achievedby sorting the composite regions of low FSC and highFL1; (3) sorting of the composite regions with both high
SSC and low–mid FL1 yielded Rhizosolenia-dominatedassemblages from the Holocene samples; (4) sorting of
high-FSC group particles yielded purified Eucampia
antarctica from the glacial samples; and (5) sorting composite regions to obtain low-FSC and low-FL1 particles

Kato et al. Progress in Earth and Planetary Science(2023) 10:13

achieved purification of needle-shaped diatoms (Thalassiothrix) from the interglacial samples.
3.5 Implications

We established a taxon-specific diatom purification
method using conventional pretreatments and a cell
sorter. The improvements in this study relative to theprevious method of Ijiri et al. (2021) are: (1) the new
method is more straightforward, with one fewer pretreatment step (omission of the elutriation process);and (2) the new method can selectively isolate a varietyof taxa, in contrast to the previous method that could
isolate only discoid diatoms.
The purification method established in this study
has many potential applications. First, it contributes
to paleoceanography, especially in high-latitude andupwelling regions where diatoms are abundant. Purification of selected diatom taxon will enable measurement of geochemical proxies such as δ18O, δ31Si, δ13C,
and δ15N of diatom frustules more accurately, removing
biases caused by differences in the ecological preferences of each taxon.
This method can also be applied to improve efficiency
in various studies involving morphological analysis of
selected diatom taxa. For example, several attempts haveused morphological variations of diatom taxa such as
Fragilariopsis kerguelensis and Eucampia antarctica as
paleoenvironmental indicators to reconstruct oceanic
fronts, sea-surface temperature, or sea-ice distribution in
the geological past (Kaczmarska et al. 1993; Cortese andGersonde 2007; Cortese et al. 2012; Allen 2014; Kloster
et al. 2018, 2019). By using the new method to enrich atarget diatom taxon and then mounting the specimens
on a microslide, the time required for microscopic observation (to find and analyze the target taxon under the
microscope) can be drastically reduced, especially when
the targeted diatom is scarce in samples.
It is also worth emphasizing that the new method successfully purifies Rhizosolenia and Thalassiothrix. Thesetaxa are the main contributors to primary/export production during summer blooming seasons in the pelagicand coastal oceans (Kemp et al. 2000, 2010); thus, further
research using the new method will yield insights into a
new aspect of biogeochemical cycles.
Moreover, the method can be extended to otherresearch fields. Possible applications include the extraction of target diatom taxa from living samples for isolating and culturing. In addition, the method would allow
non-experts to obtain rough estimations of assemblage
compositions because flow cytometric data show the relative abundances of the target particles (Fig. 4; Additional
file 2: Table S8). For example, a higher relative abundanceof Eucampia antarctica can be inferred from the higher

Page 13 of 15

density of particles in the high-FSC group (cf. R12 inFig. 4c). A prominent peak (i.e., high particle density) of
the high-FSC group in a glacial sample (Fig. 6) is consistent with the high abundance of Eucampia antarctica in
the original sample (Additional file 1: Fig. S2; Additional
file 2: Table S7).

4 Conclusions
Taxon-specific purification of diatoms is crucial for accurate geochemical analysis of diatom frustules; however,owing to the small size of diatoms, taxon-by-taxon purification has not been achieved so far. In the present study,
we developed a purification method for various diatom
taxa that uses a cell sorter. After pretreatment (removalof organic matter, carbonates, and clay minerals, followedby sieving), we conducted many cycles of sorting focusing on both optical characteristics (FSC and SSC) andfluorescence characteristics (FL1 and FL4 filters). We
selectively sorted five diatom taxa with high purity (ca.
85–100%). Specifically, (1) discoid diatoms were purified using composite regions to sort particles with bothmoderate FSC and moderate FL1 values; (2) purificationof Fragilariopsis specimens (mainly Fragilariopsis kerguelensis) was achieved by sorting the composite regionswith low FSC and high FL1; (3) sorting of the composite regions that satisfy high SSC and low–mid FL1 characteristics extracted Rhizosolenia with high purity; (4)high-FSC group particles consisted of purified Eucampia
antarctica; and (5) sorting of composite regions to obtainlow-FSC and low-FL1 particles achieved purification ofneedle-shaped diatoms (Thalassiothrix) (Figs. 5, 6, 7 andAdditional file 1: Figs. S3–S5, Additional file 2: Tables
S1–S6). Establishing a diatom purification method allows“taxon-specific analysis” in various research fields suchas paleoceanography, paleolimnology, ocean biology,
and phycology. One example of an impact of this work
is improved accuracy of geochemical analysis of diatom
frustules, leading to the advancement of paleoceanography, especially for high-latitude and upwelling regions
where diatoms are abundant.
Abbreviations
δ13C	Carbon isotopic composition
δ15N	Nitrogen isotopic composition
δ18O	Oxygen isotopic composition
δ30Si	Silicon isotopic composition
FSC	Forward scatter
IODP	International Ocean Discovery Program
JAMSTEC	Japan Agency for Marine-Earth Science and Technology
J-DESC	Japan Drilling Earth Science Consortium
mbsf	Meter below seafloor
SPT	Sodium polytungstate
SSC	Side scatter
X-Star	Institute for Extra-cutting-edge Science and Technology Avantgarde ResearchKato et al. Progress in Earth and Planetary Science(2023) 10:13

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40645-​023-​00543-5.
Additional file 1. Supplementary Figures S1–S6. Optical path of thecell sorter used in this study (Figure S1); Original diatom assemblagesof the samples (Figure S2); Flow cytograms of samples and pie charts
showing the diatom assemblages obtained from each designated region(Figure S3); Flow cytograms and pie charts/photomicrographs showing the diatom assemblages obtained by sorting of composite regions(Figure S4–S6) (PDF).
Additional file 2. Supplementary Tables S1–S8. Raw data on the diatom assemblages (Tables S1–S7); Coordinates of the designated sorting
regions (Table S8) (Excel).
Acknowledgements
We thank the officers, crew, and scientific party of cruise IODP Expedition
382 (R/V JOIDES Resolution) for sample retrieval and technical support. We
are very grateful to Ms. Momoyo Odani (Kochi University) and Ms. Harumi
Isshiki (JAMSTEC) for their technical assistance. We also thank Mr. Takeshi Izumi(Kochi University) and Dr. Fumiaki Mori (JAMSTEC) for encouraging discussion
throughout the study. This manuscript was greatly improved by constructive
comments from the reviewers.
Authors’ information
YK’s previous affiliation was Kochi University (Center for Advanced Marine Core
Research). Portions of the experiments relevant to this paper were performed
while YK was affiliated with his former institution.
Author contributions
YK conceived and designed the study, and carried out the entire experiment.
YM co-designed the study, analyzed and defined the sorting regions for thecell sorter. AI helped to interpret the data. TT supported the operation of thecell sorter. MI collaborated with the corresponding author in the constructionof the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by JSPS KAKENHI Grant Numbers 19J01441,
20H04973, and 21K14030 (YK), and 17H06318 (AI and MI). The first author(YK) also obtained a travel grant for participation in the scientific cruise IODP
Expedition 382 from the Japan Drilling Earth Science Consortium (J-DESC).
Availability of data and materials
Supporting datasets and information for this article are available in the additional files 1 and 2.

Declarations
Competing interests
The authors declare that they have no competing interest.
Received: 12 May 2022 Accepted: 21 February 2023

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