Quaternary Science Reviews 272 (2021) 107236

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Extraction method for fossil pollen grains using a cell sorter suitable
for routine 14C dating
Keitaro Yamada a, *, Takayuki Omori b, Ikuko Kitaba a, Tatsuo Hori c, Takeshi Nakagawa a, d
a
Research Centre for Palaeoclimatology, Ritsumeikan University, 1-1-1, Noji-higashi, Kusatsu, Shiga, 525-0085, Japan
b
The University Museum, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
c
Factory MARIHO, Kamakura, Kanagawa, 248-0013, Japan
d
Fukui Prefectural Varve Museum, 122-12-1, Torihama, Wakasa, Fukui, 919-1331, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Fossil pollen grains have long been recognised as potentially ideal materials for radiocarbon dating. In
Received 4 August 2021 recent years, the development of cell sorter technology has made it possible to date fossil pollen grains.
Received in revised form Although various approaches have been proposed, none of them is being practiced routinely because of
5 October 2021
the difficulty in stably and efficiently extracting small fossil grains at high enough purity from a variety of
Accepted 6 October 2021
Available online 14 October 2021
sediments. Here we show a new method which allows routine extraction and reliable radiocarbon dating
of fossil pollen grains, even from organic-rich sediments. The improved physicochemical pre-treatment
Handling Editor: Donatella Magri enables efficient enrichment from large volume of organic-rich sediments without the use of an oxidant.
We also propose new sorting criteria consisting of multi-step ‘gates’, each of which is based upon
Keywords: fluorescence characteristics and scattered light of the particles. The sorting method, combined with the
Pollen improved physicochemical pre-treatments, makes it possible to separate small fossil pollen grains from
Radiocarbon dating impurities such as green algae, micro charcoal, spores and plant fragments using a cell sorter. In
Flow cytometry particular, relatively small fossil pollen grains, with which previously proposed methods tended to suffer,
Cell sorter
could be extracted from organic-rich lacustrine sediments and organic peats in a remarkably high purity
Lake suigetsu
and velocity using our method. Radiocarbon ages subsequently measured on fossil pollen concentrates
Radiometric isotope
prepared by our method show very good agreement with those of terrestrial leaf fossils extracted from
the same sediments of Lake Suigetsu, which directly record atmospheric 14C. The extraction method
provides a new opportunity to routinely determine ages of sediments that do not contain contempo-
raneous macrofossil remains, and allow us to increase the precision of age models by strategically
selecting horizons for dating prior to incorporation into Bayesian chronological modelling.
© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction sporopollenin is a chemically inert biopolymer (Li et al., 2019) and

Fossil pollen grains theoretically offer a suitable material for
radiocarbon (14C) dating. Pollen produced on land is free from
marine reservoir effects and directly reflects the ambient atmo-
spheric 14C concentration in the same way as tree leaves, which are
typically the preferred material for 14C dating. Pollen grains are
commonplace among sedimentary deposits because pollen grains
of anemophilous plants are produced in large quantities every year
and are actively spread by wind. Fossil pollen grains consist mainly
of sporopollenin and are composed of ca. 60 wt% of carbon. Because
* Corresponding author.
E-mail address: kei-yama@fc.ritsumei.ac.jp (K. Yamada).
is resistant to diagenesis, fossil pollen grains can remain in deposits
for a very long time (even for hundreds of millions of years in
extreme cases; e.g., Books and Shaw, 1971). The nearly ‘ubiquitous’
nature of fossil pollen grains represents a significant advantage
over leaf fossils, which are usually available only very sporadically
within sediment sequences. Recent progress in chronological
modelling using Bayesian statistical methods (e.g., Bronk Ramsey,
2009) is leading to increased demand for determination of radio-
carbon ages at more numerous and strategically selected horizons.
Radiocarbon dating of pollen grains therefore offers the potential to
meet this newly emerging demand.
Despite those obvious potential advantages, however, radio-
carbon dating of fossil pollen grains has not been widely under-
taken in Quaternary science. The major obstacle for its application

https://doi.org/10.1016/j.quascirev.2021.107236
0277-3791/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
K. Yamada, T. Omori, I. Kitaba et al. Quaternary Science Reviews 272 (2021) 107236

is the difficulty to concentrate fossil pollen grains at sufficiently

high purity.
Many attempts have been made to extract fossil pollen grains for
14
C dating by physicochemical treatments modified from original
extraction methods developed for conventional palynological
analysis (e.g., Brown et al., 1989; Forster and Flenley, 1993; Regne
ll
and Everitt, 1996; Kilian et al., 2002; Piotrowska et al., 2004;
Fletcher et al., 2017), including some successful ones. However, no
de facto standard method has been established because of the
difficulties in reliably removing all impurities, such as green algae,
microcharcoal, or reworked pollen grains and spores, which have
physicochemical properties very close or similar to fossil pollen
grains. Hand picking under the microscope has also been con-
ducted as a reliable method to extract only pollen grains (e.g., Long
et al., 1992; Mensing and Southon, 1999; Schiller et al., 2021).
However, the amount of effort and time involved in such an
approach makes it unrealistic as a routine practice.
Recently, application of a ‘cell sorter’ has attracted a lot of
research attention as a new technology which has the potential to
constitute a breakthrough in high-purity extraction of fossil pollen
grains from sediments (Tennant et al., 2013; Zimmerman et al.,
2019; Kasai et al., 2021; Tunno et al., 2021). A cell sorter is a frac-
tionation device based on the technology of flow cytometry (Fig. 1).
By observing interactions (photoluminescence and scattering) be-
tween particles held in a suspension liquid and a laser beam, a cell
sorter diagnoses the particles. If the particle fulfils specific diag-
nostic criteria, then the cell sorter can recover the droplet con-
taining the target particle by pneumatic or electrostatic forces.
Sporopollenin, which is the main component of fossil pollen grains,
emits autofluorescence (e.g., Yeloff and Hunt, 2005; O'Connor et al.,
2011). Therefore, a cell sorter has the potential to diagnose and
extract fossil pollen grains without using fluorescent dyes, which
normally contain organic matter and would therefore contaminate
the samples prior to the intended radiocarbon dating. This auto-
matic picking by a cell sorter can be performed up to several
thousand times per second, allowing a cell sorter to extract fossil
pollen grains faster than manual picking.
Depending on sediment types, ages and dominant pollen taxa,
between ~103 and ~106 fossil pollen grains are needed to provide
sufficient carbon to determine radiocarbon dates with sufficiently
high precision and accuracy. Because sediments normally contain
no more than tens of thousands of grains per wet gram of sediment,
we therefore have to submit relatively large amounts of sediments
for extraction. However, it is unrealistic to introduce an even larger
volume of suspension liquid of raw sediments into the cell sorter Fig. 1. Schematic representation of cell sorter. By interacting particles in suspension
because it would simply take too long to complete the extraction. with a laser beam, the cell sorter diagnoses the particles based upon fluorescence (FL),
forward scatter (FSC) and back scatter (BSC). The sample liquid is ejected as droplets
This might even be detrimental to the cell sorter, because it is a and deflected by an electric field if it is diagnosed as containing a fossil pollen grain.
device originally developed for biosciences, comprising delicate
parts such as capillaries and nozzles, and is therefore easily jammed
when dirty liquids are introduced. Therefore, a key to the success of proven that it is possible to measure radiocarbon ages of fossil
extracting fossil pollen grains using a cell sorter is the enrichment pollen grains concentrated by cell sorter, those ages were often
of pollen prior to introduction into the cell sorter. offset from those of leaf macrofossils by several hundred years.
The extraction and dating of fossil pollen grains using a cell These methods were also tuned to a limited range of sediment
sorter was first achieved by physicochemical pre-treatment using types with no reports of successfully extracting pollen from peat,
hydrofluoric acid and nitric acid (Tennant et al., 2013). Zimmerman which is a very common material used in Quaternary science. These
et al. (2019) also succeeded in extracting and dating fossil pollen extraction methods of fossil pollen grains are, therefore, still in the
grains using a cell sorter with physicochemical pre-treatment using experimental phase and have not yet reached the stage where
nitric acid or bleach. The cell sorter itself was also improved in reliable radiocarbon ages can be routinely generated using a cell
parallel with refinement of its operation protocols. Nevertheless, sorter.
Tennant et al. (2013) and Zimmerman et al. (2019) could only The method of Zimmerman et al. (2019) has been updated by
extract pollen grains mainly with a diameter of 118 mm or less. More Tunno et al. (2021) by using more finely tuned pre-treatments.
recently, Kasai et al. (2021) established a new technology of on-chip They succeeded in preparing high-purity pollen concentrates
high-speed sorting of pollen grains using travelling vortices, from sediments of Mono Lake (CA, USA) and demonstrated that the
resulting in the purity of extracted pollen grains reaching up to 71% radiocarbon ages of the pollen concentrates were in good
on the basis of grain counts. While these pioneering studies have
2
K. Yamada, T. Omori, I. Kitaba et al.
agreement with those of plant macrofossils. Although the method
involves a filtration using 37 mm mesh, and hence cannot recover
pollen grains with a diameter of 37 mm or less, the advantages are
clearly greater than the disadvantages, at least in the case of Mono
Lake, where pollen assemblages are dominated by relatively large
pollen taxa (typically Pinaceae). Large pollen grains not only
contain more carbon per grain, but are also less likely to overlap in
size with major impurities such as green algae.
In more humid environments such as temperate and tropical
regions, however, the dominant pollen taxa are often broadleaved
trees, of which pollen grains are generally smaller. In the case of
Lake Suigetsu sediment (Japan) that we discuss later in this paper,
small pollen (<50 mm) constitutes up to 99% of the pollen assem-
blage (Nakagawa et al., 2021), and it is vital to establish an
extraction method to recover most of the small pollen grains. Pollen
concentration in organic-rich sediments, which are typical of warm
and humid regions, is often less than 10,000 grains/wet g. In order
to achieve dating of mostly small fossil pollen grains from such
sediments, it is necessary to be able to treat a 5e10 times larger
volume of sediment than in Tennant et al. (2013) and Tunno et al.
(2021).However, existing methods do not allow routine extraction
of fossil pollen grains from a large volume of organic sediment.
Moreover, oxidant treatment employed in those methods to
remove organic matter other than fossil pollen is likely to damage
the pollen grains (e.g, Jardine et al., 2015), interfering with the
further application of the methods to stable isotope analyses.
In this study, we establish a remarkably stable and efficient
method of extracting small fossil pollen grains from a large volume
of organic-rich sediments without using oxidants. The new
method, comprising a gentle pre-treatment and multi-step diag-
nostic criteria for the cell sorter, is suitable for routine generation of
radiocarbon dates. This pre-treatment method enables the
enrichment of relatively small fossil pollen grains up to 50% by
particle number, which greatly increases the efficiency and stability
of the cell sorting process. We also improved the settings of the cell
sorter, which allow us to extract fossil pollen grains at a purity of
~99% by particle number from not only lacustrine sediments but
also organic peat.
In order to validate the radiocarbon ages obtained by this new
method, we compared the 14C ages of pollen and leaves recovered
from the same organic rich sediments. For this purpose, we used
the varved sediments of Lake Suigetsu, Japan, which have the most
accurate and detailed 14C stratigraphy in the world (Bronk Ramsey
et al., 2020). The radiocarbon ages generated from the fossil pollen
grains were indistinguishable from those of the fossil leaves.
2. Material
In this study, we tested our extraction method with both
lacustrine sediments and organic peat. The lacustrine sediments
were taken from the varved sediment core obtained in 2006 from
Lake Suigetsu, Fukui Prefecture, central Japan (35
350 0800 N,
135
530 5700 E, 0 m a.s.l.) (SG06 core; Nakagawa et al., 2012). Lake
Suigetsu measures 3 km east-west by 3 km north-south, with the
surface area being approximately 4.3 km2, and a maximum water
depth of ca. 34 m. One obvious advantage of varved sediments is
that they preserve initial sedimentary structures, ensuring that
there is minimal vertical mixing of the materials. In other words,
fossils in varved sediments are quasi-exclusively preserving
contemporary carbon. Also, the SG06 core already has an excep-
tionally robust 14C stratigraphy based on >800 radiocarbon de-
terminations obtained on terrestrial leaf fossils (Staff et al., 2011;
Bronk Ramsey et al., 2012). The radiocarbon data provide the only
long, quasi-continuous, direct record of atmospheric 14C covering
the last 50 ka (Bronk Ramsey et al., 2020) and constitutes an
3
Quaternary Science Reviews 272 (2021) 107236
integral part of the international consensus radiocarbon calibration
models of IntCal13 (Reimer et al., 2013) and IntCal20 (Reimer et al.,
2020). Therefore, the varved sediment from Lake Suigetsu offers an
ideal material with which to validate a new method of radiocarbon
age determination. In this study, we took 23 samples from the
10,000e15,000 calBP (IntCal20 age scale) section of the SG06 core
(Table 1). Furthermore, we artificially homogenised the upper
100 cm of SG06 core section B-03 (spanning approximately
3700e4400 calBP; Nakagawa et al., 2012) and used it as a working
standard sample (named ‘SG06 B-03-std’). All these sedimentary
samples were deposited in a humid temperate environment and
relatively small (<50 mm) fossil pollen taxa account for 85% to
nearly 100% of the fossil pollen assemblage (Nakagawa et al., 2002,
2021).
The organic peat sample was obtained from a peat bog in
Hokkaido, Japan (42
370 N, 141
530 E, 5 m a.s.l.). This peat was
deposited in a cool temperate environment along the Atsuma River
which stretches for ca. 50 km. Moss spores and small fossil pollen
grains account for almost all of the palynomorphs. This peat was
industrially exploited by Hokkaido Peatmoss Inc. (http://www.
peatmoss.co.jp/) at Hama-Atsuma, Atsuma, Hokkaido Prefecture,
Japan (e.g., Oka et al., 2017), sieved at 6.5 mm mesh, and sold
commercially as a soil conditioner (product name: “Peat moss A
grade”). We purchased and homogenised this product and used it
as another working standard (named ‘HKD-peat-std’).
3. Physicochemical pre-treatment
Since a cell sorter measures the optical properties of every
particle, the sorting time increases proportionally to the number of
all particles introduced into the machine. The original impurities,
especially small particles such as clay minerals, which are insig-
nificant in volume but large in number, impractically hamper the
efficiency of sorting time of fossil pollen grains. Small impurities
also induce electrical clumping of particles in the sample liquid,
which cause clogging in the cell sorter.
Accordingly, physicochemical pre-treatment can decrease the
difficulty of extracting pure fossil pollen grains using the cell sorter.
The cell sorter discriminates target particles based mainly on
fluorescence. However, the difference of fluorescence intensity
between fossil pollen grains and impurities such as plant fragments
is very subtle (we cannot use organic fluorescent dye as it would be
a contaminant for the subsequent radiocarbon dating) making it
difficult to separate fossil pollen grains from other impurities at
100% accuracy using a cell sorter alone. A physicochemical pre-
treatment process selectively eliminates impurities such as plant
fragments and facilitates separation of fossil pollen grains from
other particles, by making the frequency distribution of optical
properties more clearly multimodal. In this study, we propose a
new method of relatively mild physicochemical pre-treatment,
suitable for routine extraction of fossil pollen grains by cell sorter.
It is desirable to submit a sufficient amount of carbon for high-
precision determination of ages. However, fossil pollen grains
consist of only 50e60 wt% carbon (Table 1; Omori et al. in prep). To
be on the safe side, we aimed at extracting 0.5 to 1 million grains of
fossil pollen with diameter of 10e50 mm, which is roughly equiv-
alent to 200 mg C. Assuming that samples contain ~104 fossil pollen
grains per wet gram of sediment, we therefore need to treat
30e50 g of wet sediment, which corresponds to 101e102 times the
requirement for conventional palynological analysis.
A flow chart of our pre-treatment protocol is shown in Fig. 2.
Carbonates adhere fossil pollen grains to sediments and cause
bubbling in heavy liquid separation. Firstly, we added 7 wt% HCl to
the samples and left it for 8 h at 25
C to decompose carbonates.
The treated samples were washed with distilled water and
K. Yamada, T. Omori, I. Kitaba et al. Quaternary Science Reviews 272 (2021) 107236

Table 1
Fossil pollen samples extracted from the SG06 core and their radiocarbon ages.

Lab. ID Sample definition Model age (calBP ±1s) %C mg C Conventional14C age (BP ±1s) d13C (‰)
Group Hole Section No Top(cm) Bottom(cm)

TKA-19264 SG06 B 7 180.1 182.1 11,959 ± 12 51.2 0.18 10,161 ± 49 26.11 ± 0.4
TKA-19265 SG06 B 7 185.2 187.2 12,011 ± 10 45.4 0.18 10,330 ± 52 30.10 ± 0.4
TKA-18251 SG06 A 8 164.5 166.5 12,229 ± 10 60.6 0.22 10,334 ± 89 25.20 ± 0.4
TKA-22536 SG06 B 8 5.5 7.5 12,286 ± 14 61.4 0.12 10,545 ± 65 29.07 ± 0.6
TKA-22537 SG06 B 8 13.6 15.4 12,369 ± 11 60.2 0.16 10,485 ± 54 29.66 ± 0.5
TKA-19478 SG06 A 9 22.6 24.6 12,892 ± 10 60.7 0.13 10,800 ± 42 23.51 ± 0.3
TKA-19488 SG06 A 9 24.6 26.6 12,912 ± 11 51.8 0.16 10,829 ± 50 27.41 ± 0.4
TKA-19479 SG06 A 9 28.7 30.2 12,953 ± 8 63.6 0.16 10,954 ± 55 31.48 ± 0.4
TKA-19489 SG06 A 9 35.3 37.6 13,028 ± 11 46.1 0.13 11,053 ± 45 21.93 ± 0.4
TKA-22528 SG06 A 9 37.6 39.7 13,049 ± 10 64.2 0.09 11,155 ± 85 26.72 ± 0.6
TKA-19490 SG06 A 9 39.7 41.8 13,081 ± 22 51.1 0.18 11,123 ± 47 29.46 ± 0.3
TKA-19495 SG06 A 9 46.0 48.1 13,169 ± 17 59.7 0.18 11,302 ± 46 27.69 ± 0.4
TKA-19480 SG06 A 9 54.0 56.1 13,336 ± 14 63.4 0.18 11,350 ± 45 24.92 ± 0.4
TKA-19481 SG06 A 9 56.1 58.2 13,360 ± 11 63.3 0.16 11,355 ± 56 28.26 ± 0.4
TKA-19483 SG06 A 9 58.2 60.3 13,382 ± 11 59.6 0.12 11,230 ± 50 23.43 ± 0.4
TKA-19496 SG06 A 9 60.3 62.4 13,404 ± 12 61.9 0.14 11,531 ± 59 28.51 ± 0.4
TKA-19484 SG06 A 9 60.3 62.4 13,404 ± 12 62.7 0.16 11,394 ± 48 28.56 ± 0.3
TKA-19493 SG06 A 9 66.5 68.3 13,497 ± 11 52.9 0.17 11,540 ± 43 23.01 ± 0.4
TKA-19485 SG06 A 9 68.3 70.0 13,516 ± 9 56.0 0.11 11,439 ± 49 24.10 ± 0.3
TKA-19497 SG06 A 9 70.0 71.8 13,535 ± 10 59.9 0.17 11,545 ± 50 27.16 ± 0.3
TKA-19145 SG06 B 8 123.0 125.0 13,690 ± 14 62.7 0.19 11,821 ± 62 27.16 ± 0.9
TKA-18253 SG06 A 9 95.0 97.0 13,807 ± 14 65.6 0.20 11,923 ± 122 20.70 ± 0.4
TKA-19143 SG06 B 9 14.8 16.8 14,866 ± 19 58.3 0.20 12,634 ± 57 24.66 ± 0.8

Fig. 2. Flowchart of pre-treatment proposed in this study.

centrifuged for 4 min at 1690 G (equivalent to 3000 rpm with our
facility: Model 4200, Kubota Corporation) to recover the solid
fraction. Next, we added 10 wt% KOH and heated at 90
C for 30 min
in order to remove the major part of humic acid which glues fossil
pollen grains and sedimentary particles, and/or sticks to the surface
of pollen grains. After cooling, the samples were rinsed with
distilled water five times. At this stage, some fossil pollen grains
remain in the supernatant. Therefore, we filtered the supernatant
using a 10 mm PET mesh to recover those fossil pollen grains. This
fraction was put aside until it was re-combined with the main
sample in a later stage.
The main sample after rinsing was subjected to a 10 wt% HCl
treatment at ambient room temperature to acidify the liquid. Then,
we mixed the sample with a solution of zinc chloride and centri-
fuged at 1690 G (equivalent to 3000 rpm in our lab) for 30 min. The
density of the heavy liquid was adjusted between 1.88 g/ml (typi-
cally suitable for lacustrine sediments; Nakagawa et al., 1998) and
1.78 g/ml (samples rich in organic debris such as peat) to maximize
the recovery of fossil pollen grains. The previously noted particles
filtered from the rinsing water were mixed with the light fraction
4
after the heavy liquid separation.
The mixed samples were sieved with a 50 mm mesh to remove
the large fraction that may fall into the target size fractions
(410e50 mm) through subsequent chemical treatments. The sieved
samples (<4 50 mm) were mixed with 10 wt% KOH and heated at
90
C for 20 min for further removal of impurities such as plant
fragments. We restricted the heating time to less than 1 h in total
because it would otherwise become difficult to precipitate fossil
pollen grains due to static electricity induced by the alkali treat-
ment. We then sieved the samples for one final time at 10 mm to
remove all impurities newly produced by the chemical stresses of
the preceding KOH treatments. Using our facilities, we can typically
treat 4e8 samples per person per day following this protocol.
4. Cell sorter extraction
In this study, we used an SH800Z cell sorter (SONY, Tokyo,
Japan) of which sample lines consist exclusively of disposable parts.
This made it significantly easier and faster to exchange sample lines
if they became clogged by sedimentary particles, which cell sorters
K. Yamada, T. Omori, I. Kitaba et al. Quaternary Science Reviews 272 (2021) 107236

were not originally designed for.
Fossil pollen grains treated by KOH easily swell up with water.
We repeatedly sieved the samples with a 50 mm mesh to avoid
jamming the capillary tube of sample lines by swollen pollen grains
before introduction into the cell sorter. Moreover, particles in pre-
treated samples tend to clump together electrostatically,
enhancing the likelihood of clogging. To avoid the flocculation of
particles, we turned the sample suspension liquid basic by adding
the minimum required volume of KOH.
We used 0.8%(w/w) saline solution, without any organic sur-
factant, for the sheath water. All introduced particles were excited
by a laser collinear beam consisting of multiple laser beams of
405 nm and 488 nm and are detected as ‘events’. In this study, we
observed fluorescence intensities at wave-length channels of
450 ± 25 nm, 525 ± 25 nm, 600 ± 30 nm, 665 ± 15 nm, 720 ± 30 nm
and 785 ± 30 nm, as well as forward scatter (FSC-A, FSC-W, FSC-H)
and backscatter (SSC) intensities, of every particle.
The observed properties of particles in a sample are immedi-
ately expressed as dots in a scatter plot in a 2D space (defined by
two selected optical properties), each dot corresponding to a single
particle, colour-coded by data density per area in the 2D space
(Fig. 3aed). Particles with different optical properties make clusters
in this scatter diagram. Looking at such a clustering pattern, the cell
sorter operator can define a geometric area in the space called a
‘physical gate’. The particles that fall into this physical gate are
either further examined using another 2D scatter diagram defined
by a different pair of optical properties, or eventually recovered in a
sample tube, if the target particle is most likely pollen. In this study,
we defined each physical gate with ellipses because the probability
density distribution of each cluster in the sediments is supposed to
show a logarithmic-normal distribution (Fig. 3aed). We designed a
multilevel ‘Boolean gate’ that consists of a series of physical gates
for extracting fossil pollen grains as below. Here we explain the
most typical Boolean gate with which we could successfully extract
fossil pollen grains from the varved sediments obtained from Lake
Suigetsu (SG06 B-03-std).
Firstly, we designed Gate P1 on the scatter graph plotted by FSC-
W and 785 ± 25 nm in order to remove clearly too small and low
fluorescing particles such as plant fragments (Fig. 3a and e, Gate
O1) and clay minerals (Fig. 3a, Gate O2). The particles selected by
Gate P1 were characterised by a strong fluorescence intensity of
785 ± 25 nm and consisted of about 60% fossil pollen grains (by
number of particles) (Fig. 3a). Next, we submitted the cluster
filtered by Gate P1 to the next scatter plot defined by 450 ± 25 nm
and 525 ± 25 nm for the purpose of removing spores and plankton
(Fig. 3b). On this diagram, two obvious clusters were recognised,
which we defined with three corresponding gates P2-1 and P2-2.
While the clusters specified by Gates P2-1 (Fig. 3f) and P2-2
(Fig. 3g) consisted of >80% (by particle number) fossil pollen
grains, all clusters still contained irregular particles such as plant

Fig. 3. Optical properties of particles in the SG06 B-03-std after pre-treatment, measured by cell sorter. The colour of each dot represents the density of particles with similar
properties (aed). The regions designated by ellipses are called ‘gate’. (a): Firstly, the properties of all particles are plotted by FSC-W and 785 ± 25 nm. The particles that fell into the
Gates P1, O1 and O2 are mainly composed of fossil pollen grains (sent to subsequent gates), plant fragments including green algae (Fig. 3e), and minerals, respectively. (b): Secondly,
the particles that have passed through Gate P1 are plotted by fluorescence in 450 ± 25 nm and 525 ± 25 nm. Fossil pollen grains concentrate in the regions defined by Gate P2-1
(Fig. 3f; mainly consisting of Quercus) and Gate P2-2 (Fig. 3g; mainly consisting of Cryptomeria) which are subsets of the preceding Gate P1. (c), (d): Finally, the particles defined by
Gate P2-1 and Gate P2-2 are plotted by fluorescence in 525 ± 25 nm and 600 ± 30 nm. The particles that passed through Gates P3-1 and P3-2 were recovered and combined
(Fig. 3h). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

5
K. Yamada, T. Omori, I. Kitaba et al. Quaternary Science Reviews 272 (2021) 107236

fragments and plankton. We, therefore, submitted clusters P2-1

and P2-2 to further Gates 3e1 and 3e2, defined in the space of
525 ± 25 nm and 600 ± 30 nm, to eliminate those irregular particles
which, in this scatter, have much larger deviations from the mean
than fossil pollen grains (Fig. 3c and d). Clusters P3-1 and P3-2
consisted of 99% fossil pollen grains by particle number (Fig. 3h).
When the sample is particularly rich in organic matter (which was
the case with the HKD-peat-std), the samples were further filtered
by an additional gate in a scatter diagram defined by the intensities
of 665 ± 15 nm and 720 ± 30 nm.
After diagnosis by the Boolean gate, the sheath liquid including
particles with measured optical properties were ejected at a rate of
10,000 droplets per second from a nozzle. When a droplet contains
a fossil pollen grain as diagnosed by the Boolean gate, the cell sorter
electrically charges the droplet and deflects the droplet with an
electric field (Fig. 1). In reality, droplets sometimes contain not only
one grain of fossil pollen but also impurities and/or multiple pollen
grains. In this study, we prioritised purity even at the cost of re-
covery rate, and hence rejected any droplets that contained not
only fossil pollen grains but also any other materials. Here, the
number of sorted particles relative to the number of all detected
particles is called ‘sort efficiency’. Sort efficiency can be improved
by diluting the sample suspension and reducing the rate of ‘events’.
We achieved efficient extraction of fossil pollen grains without
sacrificing the recovery rate by diluting the sample suspension
liquid until the sort efficiency exceeded 90%.
The forming of droplets is affected by the viscosity of the sheath
water, which is a function of the temperature. While the cell sorter
used in this study automatically adjusts the shape of the droplets to
account for temperature changes, it becomes difficult to maintain
the shape when the rate of temperature change exceeds a certain
Fig. 4. Changes of event rates detected by the cell sorter with (a) and without (b) KOH
level. We controlled the room temperature change to within added to the sample suspension liquid when sorting. (a): The event rate is stable for an
±0.5
C to stabilise the formation of droplets. In this study, we hour, which means that the cell sorter is not clogging with the particles. The decrease
collected 0.5 to 1 million fossil pollen grains from each sediment of the event rate is negligible (0.1 eps/min), showing that the particles stayed ho-
sample. The extraction was performed at the Research Centre for mogeneously dispersed in the liquid for an hour. (b): The event rate showed large
variance with occasional sharp decreases due to clogging. The event rate also steadily
Palaeoclimatology, Ritsumeikan University. The radiocarbon ages of
decreased (1.7 eps/min), meaning that the concentration of the introduced particles
extracted fossil pollen concentrates were subsequently measured changed by ca. 14% in an hour.
by accelerator mass spectrometry (AMS) in the University Museum,
University of Tokyo (Omori et al. in prep).
pollen grains in the sample prior to sorting are cemented by im-
5. Results & discussion purities (Fig. 6a and b), whereas the sample after sorting is visibly
very clean (Fig. 6c and d). The fossil pollen grains extracted by the
(1) Extraction of fossil pollen using the cell sorter cell sorter are still intact and preserve original surface ornamen-
tations and forms. This provides support for the physicochemical
Two examples of time-series changes of event rate detected by pre-treatments not causing significant loss of target materials.
the cell sorter, with and without application of the KOH dispersant,
are shown in Fig. 4. The one without KOH showed a gradual (2) Purification and efficiency in sorting
decrease of event rate from ca. 700 to ca. 600 in 1 h with a little
clogging (Fig. 4b), indicating aggregation of the particles. In The fluorescence spectra of particles that are recovered by the
contrast, the sample with KOH showed a quasi-constant event rate gates P3-1, P3-2, O1 and O2 (Fig. 3) are shown in Fig. 7. The particles
of 426 ± 43.5 events per second (eps) (Fig. 4a), showing that the designated by Gate P3-1 and P3-2 consisted mainly of fossil pollen
particles did not aggregate in the tube. The aggregation of sample grains. The pollen grains recovered by Gate P3-1 were composed
particles inevitably reduces sort efficiency of fossil pollen grains mainly of Quercus section Cyclobalanopsis, Castanea-Castanopsis,
and may even cause clogging of the cell sorter in the worst cases. and those by Gate P3-2 were composed mainly of Cryptomeria,
The stable operation of the cell sorter without clogging is one of the Taxaceae-Cupressaceae. The grains recovered by Gates O1 and O2
most critical factors to control purity and time efficiency of the were mainly plant fragments (including green algae) and minerals,
sorting, especially when the required number of sorted particles is respectively.
large (which is the case for the radiocarbon dating of pollen). Although both fossil pollen grains and plant fragments emit
The pre-treated sample of the SG06 B-03-std prior to sorting autofluorescence at 525e720 nm, fossil pollen grains fluoresce
contains visible impurities such as small mineral particles of a few more strongly than plant fragments. The intensity of fluorescence
mm, black microcharcoals and semi-transparent plant fragments of a fossil pollen grain is strongly correlated with the amount of
(Fig. 5b). In contrast, the sample extracted by the cell sorter consists sporopollenin. In this study, we observed strong fluorescence at
almost exclusively of fossil pollen grains and reaches a remarkably 525 ± 25 and 600 ± 30 nm. The fossil pollen grains recovered by
high purity of 99% by grain count (Fig. 5c). Gates 3e1 and 3e2 contained different taxonomic groups and
Fig. 6 shows electron micrographs of the sorted materials. The showed different patterns of fluorescence (Fig. 7) even though the
6
K. Yamada, T. Omori, I. Kitaba et al.
Fig. 5. Micrographs of the lake sediment (SG06 B-03-std) at different preparation stages (a, b, c) and organic peat (HKD-peat-std; d). (a): Smear image. (b): After pre-treatment. The
concentrate is mainly composed of pollen grains, green algae and mineral particles. (c), (d): End products after cell sorter obtained from SG06 B-03-std and HKD-peat-std,
respectively. The purity of fossil pollen grains finally reached 99% by grain count. (For interpretation of the references to colour in this figure legend, the reader is referred to
the Web version of this article.)
grains were obtained from the same sample. Thus, the intensity of
fluorescence measured by the cell sorter is likely to also be affected
by the structures of pollen grains such as thickness and supra-
sculptures, implying the potential to distinguish different pollen
types using fluorescence (O'Connor et al., 2011). These optical
properties also allow separation of fossil pollen grains and other
impurities which have similar physicochemical properties, such as
green algae and micro charcoal.
The cell sorter can also distinguish between fossil pollen and
spores even though both are composed of the same material,
sporopollenin. The fluorescence patterns of fossil pollen grains and
spores detected by the cell sorter are shown in Fig. 8. Though the
fossil spores emit stronger fluorescence than organic debris, the
absolute level of the fluorescence of spores is about half of that of
pollen grains. It is assumed that the doubled signal intensity of
pollen occurs because fossil pollen has a double wall structure
while the fossil spore has a single wall. The cell sorter opens up a
new opportunity to eliminate spores from pollen concentrates. This
is a significant advantage of the protocol that we propose because
the radiocarbon ages of spore fossils can potentially be affected by
relatively old carbon in soil.
The cell sorter may also be able to remove reworked pollen
grains. The fluorescence properties are known to change with
diagenesis (e.g., Strother et al., 2017). In the case of the Lake Sui-
getsu sediments, the fluorescence intensity of typical pollen clus-
ters tended to shift according to their ages; i.e., the elliptical gates
shown in Fig. 3 only indicate relative regions within the scatter and
their absolute coordinates change according to their ages. This
strongly implies that reworked pollen grains (if there are any) are
more likely not to fall within the gates that are optimised for the
7
Quaternary Science Reviews 272 (2021) 107236
fossil pollen grains contemporaneous to the sediment.
Some types of plant fragments are difficult to distinguish from
fossil pollen grains based only upon fluorescence. Because the
probability distributions of fluorescence properties of fossil pollen
grains and plant fragments are close to each other, the tails of their
distributions overlap. Thus, if we are to reduce such particles, then
we would have to abandon a considerable proportion of fossil
pollen grains. Instead of narrowing gates any further, in this study,
we performed a second KOH stage in the pre-treatment to mini-
mise the overlap of the distributions (Fig. 2) and achieved 99%
purity of pollen. The elimination of impurities by the second KOH
treatment (Fig. 2) is especially effective for organic peat samples.
KOH also enhances the fluorescence of pollen membranes and
makes the division between fossil pollen grains and other impu-
rities clearer (Fig. 9).
Moreover, we succeeded in improving the sorting time and the
‘sort efficiency’ considerably by reducing particles of <10 mm by the
pre-treatment. The clay size particles defined by the complement of
Gate O2, which mainly consist of minerals and plant fragments
reduced by the second KOH treatment, are characterised by almost
no fluorescence and low FSC. Because the cell sorter measures all
particles (regardless of size) and the particles need to be thinly
diluted, the presence of small particles (and consequent dilution of
the sample suspension liquid) directly increases sorting time. The
small particles also reduce the sort efficiency down to <80% by
particle number, probably due to electrostatic aggregation with
fossil pollen grains. In this study, we removed the small particles
using a 10 mm mesh. Though the ‘small particles’ may include some
pollen, such as Castanopsis, the proportion of such pollen taxa in the
pollen assemblage is generally low and, hence, the total amount of
K. Yamada, T. Omori, I. Kitaba et al.
Fig. 6. Electron micrographs of the pre-treated lake sediments (SG06 B-03-std) before (a, b) and after (c, d) purification by the cell sorter. (a), (b): The samples are rich in fossil
pollen grains but still contain fragments of green algae and minerals. (c), (d):The samples are almost pure in fossil pollen grains with a variety of shapes and surface ornamentations.
Fig. 7. The fluorescence spectra of the clusters specified by different gates. The fluo-
rescence was measured in 6 channels of wavelength (450 ± 25, 525 ± 25, 600 ± 30,
665 ± 15, 720 ± 30 and 785 ± 30 nm) and is shown with ±1s error bars. The most
typical particle types are noted in parentheses. The pollen-rich fraction defined by
Gates P3-1 and P3-2 have stronger fluorescence than other clusters rich in plant
fragments (Gate O1) and minerals (Gate O2). The pollen grains extracted by Gate P3-1
are generally larger and have more complex surface structures than those of Gate P3-2.
carbon contained in such ‘small’ pollen grains is normally pre-
sumed to be negligible.
(3) Extracting bias for pollen assemblage
The extracted pollen grains consisted of multiple pollen taxa
8
Quaternary Science Reviews 272 (2021) 107236
Fig. 8. The fluorescence spectra of fossil pollen grains and spores in the organic peat
(HKD-peat-std) with ±1serror bars. The fluorescence is measured in 6 channels
(450 ± 25, 525 ± 25, 600 ± 30, 665 ± 15, 720 ± 30 and 785 ± 30 nm) by the cell sorter.
Fossil pollen grains have a double wall structure and thus fluoresce about twice as
strongly as fossil spores which only have a single wall.
and the major pollen flora with a diameter of <50 mm did not
change significantly as compared to the pre-sorted pollen abun-
dances. The pollen spectra of concentrates are shown in Fig. 10. The
samples after cell sorter extraction were composed of the taxa with
a diameter of <50 mm, including Quercus Cyclobalanopsis, Carpinus,
Alnus, Fagus, and Cryptomeria (Fig. 10b and d). In contrast, the taxa
with a diameter of >50 mm, such as Pinus, are understandably
biased by the 50 mm mesh fractionation before sorting. Because
pollen grains with a diameter of >50 mm are usually produced by
K. Yamada, T. Omori, I. Kitaba et al. Quaternary Science Reviews 272 (2021) 107236

Fig. 9. Optical properties of the SG06 B-03-std introduced to the cell sorter without (a) and with KOH (b) when sorting. The change in pH caused by the addition of KOH not only
ensures a stable dispersal of grains in the liquid but also makes the fluorescence of fossil pollen grains more pronounced, making it easier to separate fossil pollen grains by the cell
sorter.

Fig. 10. The pollen taxa composition of the samples of lake sediments (SG06 B-03-std; a, b) and organic peat (HKD-peat-std; c, d) at different steps of the preparation. (a), (c): before
extraction by the cell sorter (CS). (b), (d): after extraction by the CS. The pollen flora with a diameter of <50 mm, including Quercus Cyclobalanopsis, Carpinus, Alnus, Fagus, and
Cryptomeria, did not change significantly.

conifers, the effect of size bias in this method is limited in envi-
ronments where broad leaf trees dominate and few conifers are
distributed.
The percentage taxa composition of pollen changed after sorting
(Fig. 10). Pollen with either a smooth surface, such as Taxaceae-
Cupressaceae, or particularly complex structures such as Ulms-
Zelkova type are selectively lost using the cell sorter. These taxa are
supposed to have weaker or stronger fluorescence in comparison to
other genera, due to lower or higher density of sporopollenin per
unit area. This is also in line with the observation that the fluo-
rescence level of modern pollen differs according to the taxonomic
group (e.g., O'Connor et al., 2011). Although it is beyond the scope of
this paper, the cell sorter may have the power to separate pollen
grains of different taxonomic groups based on optical properties,
which will be the subject of future research.
(4) Radiocarbon dating of fossil pollen
We validated the radiocarbon ages of fossil pollen grains by
comparing with those of the leaf macro fossils obtained from the
same varved sediment core (SG06) from Lake Suigetsu (Fig. 11). The
agreement between radiocarbon ages of pollen and leaves is
generally very good, demonstrating no systematic offset. We
therefore conclude that our pollen extraction method is no longer
just experimental, but is indeed applicable to routine analysis of a
wider range of organic-rich materials such as lacustrine sediments
and peat, when intact terrigenic plant macro fossils ideal for
radiocarbon dating are not available.
The extracted fossil pollen grains were subsequently subjected
to routine acid-alkali-acid (AAA) treatment for radiocarbon dating.
This post-treatment was indispensable for the removal of humic

9
K. Yamada, T. Omori, I. Kitaba et al.
Fig. 11. Radiocarbon ages of the fossil pollen grains and the leaf macro fossils obtained
from Lake Suigetsu. The ages of the fossil pollen are consistently in good agreement
with those of the leaf fossils.
acid adhering to the surface of pollen grains (Omori et al. in prep.).
We consumed ca. 1/2 million fossil pollen grains for each mea-
surement, which corresponds to 100e200 mg C (Table 1). The fossil
pollen grains contained 50e60 wt% of carbon, which is in concor-
dance with previous reports (e.g., Piotrowska et al., 2004).
The radiocarbon ages of the fossil pollen grains seem to be
showing even less variation than those of the leaves. This might be
because the radiocarbon ages of the fossil leaves from Lake Suigetsu
are snapshots of a specific season in a single year (the leaf fossils
mainly derive from deciduous trees). In contrast, it is considered
that the radiocarbon ages of the fossil pollen grains typically reflect
the average atmospheric 14C concentration over the 20e30 year
sampling interval represented by the 2 cm thick intervals of the
core from which the fossil pollen grains were extracted.
Some pioneer studies of radiocarbon dating of fossil pollen
grains suffered from suboptimal agreement with macro fossil re-
mains (e.g., Zimmerman et al., 2019; Kasai et al., 2021), which was
(fortunately) not the case with the present study. It is considered
that the causes of such disagreement include insufficient purity of
pollen samples, and/or contamination by modern atmospheric CO2.
Further details of the radiocarbon dating of the fossil pollen grains
are discussed by Omori et al. (in prep.).
6. Concluding remarks
Small fossil pollen grains can be routinely extracted from
organic-rich lacustrine sediments and organic peat by the com-
bined method of our physiochemical pre-treatment and subse-
quent purification with a cell sorter. The purity of the extracted
pollen exceeded 99% in all cases tested in this study, strongly
implying that the method can be applied to a wider range of
organic-rich materials such as lacustrine sediment and peat. The
radiocarbon ages measured on the pollen concentrates extracted by
our method showed very good agreement with those measured on
the fossil leaves from the same SG06 core from Lake Suigetsu, Japan.
Because fossil pollen grains are more robust than fossil leaves and
distributed more universally in a variety of sediments, the new
method enables us to determine radiocarbon ages at numerous and
10
Quaternary Science Reviews 272 (2021) 107236
strategically selected horizons, which is ideal for combination with
Bayesian statistical modelling approaches to age determination
(Bronk Ramsey, 2009). The application of the method to well age-
constrained varved sediment cores from Lake Suigetsu may also
contribute to the international consensus radiocarbon calibration
models which require precise, accurate and numerous radiocarbon
determinations.
Author contributions
Keitaro, Yamada: Investigation, Methodology, Writing e orig-
inal draft, Funding acquisition Takayuki Omori: Investigation,
Methodology, Writing e review & editing Ikuko Kitaba: Investi-
gation, Writing e review & editing, Funding acquisition Tatsuo
Hori: Software, Methodology, Writing e review & editing Takeshi
Nakagawa: Investigation, Project administration, Supervision,
Conceptualization, Writing e review & editing, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This study was supported by KAKENHI grants from the Japan
Society for the Promotion of Science [grant numbers 16K13894,
18H03744, 19K20442 and 19H04256], the Sumitomo Foundation
Grant for Environmental Research Projects, Japan [grant number
194020], the Casio Science Promotion Foundation fund, Japan
[grant number 35-06], and by the Ito Foundation for Science, Japan.
We are grateful to the Fukui Prefectural Varve Museum for
providing the cell sorter. This study was partly conducted as a joint
research project with Kansai Electric Power Co., Inc. We thank Dr. D.
Ishimura for providing the early generation of test sedimentary
samples. We also thank Dr. R. A. Staff for careful proofreading of the
manuscript and helpful comments, and Mr. A. Yamasaki for mixing
the HKD-peat-std. Finally, we wish to express our sincere gratitude
to Dr. Susan R H Zimmerman and another anonymous reviewer for
useful comments and improving the manuscript.
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