Confocal measurement of microplastics uptake by plants

Abstract

Microplastics (MPs, plastics 100 nm-5 mm in diameter) are estimated to accumulate in agricultural soils in quantities that exceed the total MP burden in ocean waters. Despite a wealth of information relating to the accumulation of MPs in aquatic species, there is little information on the uptake of MPs by terrestrial plants. Information about location of MPs in plant tissues is critical to understand the modes of their interaction with plants. Polystyrene (PS) is one of the most commonly used plastic polymers worldwide and it is often found in MPs sampled in the environment. The performance of traditional detection methods (i.e., transmission electron microscopy, TEM and scanning electron microscopy, SEM) for nanoparticles is limited due to the extensive sample preparation and the limited field of view. Here we report an approach for the imaging of different sizes of PS plastic beads (ranging from submicrometer to micrometer-sized) within plant tissues by using confocal laser scanning microscope (CLSM). Fluorescent dye Nile blue or 4-chloro-7-nitro-1,2,3-benzoxadiazole were encapsulated into the PS microbeads through swelling method and they were used to detect the localization of PS beads in the root and the green tissue respectively. •This is a simple and rapid approach for imaging of MPs in plant.•The fluorescent dyes can produce bright and stable emission signals that are distinguishable from the autofluorescence background of plant tissues.•The dyes leakage in the aqueous phase can be assumed to be negligible.

Full text

Method
Article
Confocal
measurement
of
microplastics
uptake
by
plants
Lianzhen
Li
a
,
Yongming
Luo
a,b,
*,
Willie
J.G.M.
Peijnenburg
c,d
,
Ruijie
Li
a
,
Jie
Yang
b
,
Qian
Zhou
a
a
Key
Laboratory
of
Coastal
Zone
Environmental
Processes
and
Ecological
Remediation,
Yantai
Institute
of
Coastal
Zone
Research
(YIC),
Chinese
Academy
of
Sciences
(CAS),
China
b
Key
Laboratory
of
Soil
Environment
and
Pollution
Remediation,
Institute
of
Soil
Science
(ISSAS),
Chinese
Academy
of
Sciences
(CAS),
China
c
National
Institute
of
Public
Health
and
the
Environment,
Center
for
Safety
of
Substances
and
Products,
P.O.
Box
1,
3720
BA,
Bilthoven,
the
Netherlands
d
Institute
of
Environmental
Sciences
(CML),
Leiden
University,
Leiden,
the
Netherlands
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Accepted
21
November
2019
Available
online
3
December
2019
Method
name:
A
simple
and
rapid
approach
for
imaging
of
microplastics
in
plant
Keywords:
Microplastics
Confocal
laser
scanning
microscopy
Plant
uptake
Fluorescent
imaging
A
B
S
T
R
A
C
T
Microplastics
(MPs,
plastics
100
nm–5
mm
in
diameter)
are
estimated
to
accumulate
in
agricultural
soils
in
quantities
that
exceed
the
total
MP
burden
in
ocean
waters.
Despite
a
wealth
of
information
relating
to
the
accumulation
of
MPs
in
aquatic
species,
there
is
little
information
on
the
uptake
of
MPs
by
terrestrial
plants.
Information
about
location
of
MPs
in
plant
tissues
is
critical
to
understand
the
modes
of
their
interaction
with
plants.
Polystyrene
(PS)
is
one
of
the
most
commonly
used
plastic
polymers
worldwide
and
it
is
often
found
in
MPs
sampled
in
the
environment.
The
performance
of
traditional
detection
methods
(i.e.,
transmission
electron
microscopy,
TEM
and
scanning
electron
microscopy,
SEM)
for
nanoparticles
is
limited
due
to
the
extensive
sample
prepara-
tion
and
the
limited
field
of
view.
Here
we
report
an
approach
for
the
imaging
of
different
sizes
of
PS
plastic
beads
(ranging
from
submicrometer
to
micrometer-sized)
within
plant
tissues
by
using
confocal
laser
scanning
microscope
(CLSM).
Fluorescent
dye
Nile
blue
or
4-chloro-7-nitro-1,2,3-benzoxadiazole
were
encapsulated
into
the
PS
microbeads
through
swelling
method
and
they
were
used
to
detect
the
localization
of
PS
beads
in
the
root
and
the
green
tissue
respectively.
*
Corresponding
author
at:
Key
Laboratory
of
Coastal
Zone
Environmental
Processes
and
Ecological
Remediation,
Yantai
Institute
of
Coastal
Zone
Research
(YIC),
Chinese
Academy
of
Sciences
(CAS),
China.
E-mail
address:
ymluo@issas.ac.cn
(Y.
Luo).
https://doi.org/10.1016/j.mex.2019.11.023
2215-0161/©
2019
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY
license
(http://
creativecommons.org/licenses/by/4.0/).
MethodsX
7
(2020)
100750
Contents
lists
available
at
ScienceDirect
MethodsX
journal
homepage:
www.elsevier.com/locate/mex


This
is
a
simple
and
rapid
approach
for
imaging
of
MPs
in
plant.

The
fluorescent
dyes
can
produce
bright
and
stable
emission
signals
that
are
distinguishable
from
the
autofluorescence
background
of
plant
tissues.

The
dyes
leakage
in
the
aqueous
phase
can
be
assumed
to
be
negligible.
©
2019
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.
org/licenses/by/4.0/).
Specification
Table
Subject
Area:
Environmental
Science
More
specific
subject
area:
Environmental
Impact/Environmental
Ecotoxicology
Method
name:
A
simple
and
rapid
approach
for
imaging
of
microplastics
in
plant
Name
and
reference
of
original
method:
NO
reference
Resource
availability:
No
resource
available
Method
details
Synthesis
and
characterization
of
fluorescently
labeled
polystyrene
particles
Fluorescent
PS
particles
(without
functionalization)
were
customized
synthesized
from
Da'e
Scientific
Co.,
Ltd.
(Tianjin,
China).
The
two
sizes
of
PS
microbeads
were
first
synthesized
by
means
of
dispersion
polymerization
and
a mini-emulsion polymerization process [1]. Then,
the fluorescent
dye Nile
blue
(NB) or
4-chloro-7-nitro-1,2,3-benzoxadiazole
(NBD-Cl)
was
immobilized
into
the
polymer
matrix
through
a
swelling
method
by
dispersing
PS
beads
in
0.25
%
sodium
dodecyl
sulfate
(SDS)
and
taking
methylene
dichloride
as
swelling
agent
[2].
The
labeled
beads
were
centrifuged
and
washed
with
ethanol
and
deionized water toremove any free dyes until there is no fluorescencedetected in the supernatant. The stock
solutions
were
supplied
as
10
mg
mL
1
suspensions
in
ultrapure
water
(18.2
M
V
,
Millipore,
USA).
The
PS
beads
had
primary
nominal
sizes
of
0.2
m
m
and
2.0
m
m.
The
actual
sizes
of
the
0.2
m
m
and
2.0
m
m
PS
microbeads
were
determined
to
be
0.23

0.04
m
m
and
1.9 8

0.08
m
m
using
SEM
(Fig.
S1).
These
two
fluorescent
dyes
were
used
to
detect
the
presence
of
PS
beads
because
the
red
fluorescence
from
NB
is
indistinguishable
from
the
red
autofluorescence
emitted
by
chloroplasts
and
the
green
fluorescence
from
NBD-Cl
is
indistinguishable
from
the
green
autofluorescence
in
root
tissues
(Fig.
S2).
Thus,
in
this
study
PS-NB
was
used
to
detect
PS
beads
in
roots,
and
PS-NBD-Cl
was
used
to
detect
PS
beads
in
stems
and
leaves.
To
assess
the
stability
of
the
fluorescence,
we
analyzed
the
loss
of
fluorescence
intensity
after
exposure
of
the
PS
beads
in
Hoagland
solutions
within
plants
for
3,
6,
12,
24,
48,
and
72
h
using
a
Synergy
H1
microplate
reader
(Bio
Tek,
Winooski,
VT,
USA)
with
excitation
and
emission
wavelengths
of
488
nm
and
518
nm
or
635
nm
and
680
nm.
The
leakage
of
fluorescence
was
assessed
by
measuring
the
fluorescence
before
and
after
filtration
through
a
0.1-
m
m
syringe
filter
at
the
end
of
the
indicated
incubation
periods.
For
Nile
Blue
labeled
0.2
m
m
and
2.0
m
m
PS
beads
suspensions,
the
amount
of
dye
after
filtration
was
below
to
the
microplate
reader
detection
limit.
The
dissolved
dye
4-chloro-7-nitro-1,
2,
3-benzoxadiazole
release
into
the
solution
did
not
exceed
3.7
%
for
the
PS
beads
(Fig.
S3).
Consequently,
the
presence
of
fluorescent
markers
in
the
aqueous
phase
can
be
assumed
to
be
quite
negligible.
The
average
hydrodynamic
diameter
and
the
zeta
potential
of
the
PS
beads
in
Hoagland
solution
were
determined
by
dynamic
laser
scattering
(DLS)
using
a
Malvern
Zetasizer
Nano-ZS90
(ZEN3590,
UK).
Both
stock
suspensions
were
sonicated
for
2
min
using
a
sonicator
(Branson
Ultrasonic,
Danbury,
CT,
USA)
and
2
L.
Li
et
al.
/
MethodsX
7
(2020)
100750

were
then
diluted
to
obtain
the
final
concentrations
of
the
beads
used
in
the
tests.
The
average
PS
beads
hydrodynamic
diameter
was
0.27

0.04
m
m
and
2.5

0.3
m
m
for
the
0.2-
m
m
and
2.0-
m
m
PS
beads,
respectively.
The
surface
of
the
MPs
was
negatively
charged,
with
the
0.2-
m
m
beads
having
a
more
negative
zeta
potential
(42.7

0.9
mV)
than
the
2.0-
m
m
beads
(12.1

0.6
mV).
Plant
materials
and
growth
conditions
Wheat
was
chosen
as
a
model
plant
in
this
study
because
of
its
wide
production
as
one
of
the
main
world-wide
foodstuffs
in
the
world.
Seeds
of
wheat
(Triticum
aestivum
L.,
Zhongmai
9)
were
supplied
by
the
Chinese
Academy
of
Agricultural
Sciences,
Beijing,
China.
Wheat
seeds
of
an
almost
uniform
size
were
selected
and
surface
sterilized
by
treatment
with
a
10
%
NaClO
solution
for
5
min.
Subsequently,
the
seeds
were
washed
three
times
with
deionized
water
to
remove
the
residual
NaClO
solution
and
were
then
transferred
to
moistened
filter
paper
and
incubated
in
the
dark
at
25

C
to
induce
germination.
After
four
days,
four
seedlings
with
uniform
size
were
transferred
to
a
1-L
beaker
containing
Hoagland
solution.
The
beaker
was
covered
with
a
cap
and
placed
in
a
growth
chamber
(illumination
of
250
m
mol
m
2
s
1
for
a
12
h/12
h
day/night
photoperiod;
70–80
%
relative
humidity;
25

2

C).
Each
box
was
equipped
with
an
aquarium
aerator
to
provide
oxygen
to
the
roots
and
to
maintain
the
PS
beads
in
suspension.
The
wheat
plants
were
exposed
to
PS
microbeads
for
10
days.
The
experiment
was
repeated
three
times.
Imaging
by
confocal
laser
scanning
microscopy
Following
exposure
to
PS
beads,
the
roots
were
removed
and
washed
thoroughly
with
distilled
water.
Subsequently,
fresh
root
(mature
zone)
and
stem
(2
cm
from
the
base
of
the
stem)
segments
were
collected
and
embedded
in
4
%
agarose.
The
root
and
stem
samples
and
the
leaf
blades
(with
the
primary
vein)
were
sectioned
into
40-
and
100 -
m
m
thick
sections,
respectively,
using
a
vibrating
microtome
(VT1200S
Vibrotome,
Leica,
Vienna).
Semi-thin
sections
of
the
samples
were
placed
on
a
glass
slide
covered
with
a
coverslip,
and
a
few
drops
of
PBS
were
added
to
keep
the
sample
hydrated.
The
sectioned
tissue
was
inspected
using
a
confocal
laser
scanning
microscope
(FluoView
FV1000;
Olympus,
Japan)
with
excitation/emission
wavelengths
of
488/515
nm
and
635/680
nm
for
NBD-Cl
and
NB,
respectively.
Transverse
and
longitudinal
sections
of
at
least
three
plants
were
examined
from
each
treatment
group
before
representative
images
were
selected.
Images
were
captured
with
a
SPOT
camera
and
imported
into
imaging
software
Fluoview
FV
10
for
the
compound
microscope.
Pictures
have
brightness
and
contrast
enhancement.
Observation
by
scanning
electron
microscopy
Selected
samples
from
the
roots,
stems
and
fully
expanded
leaves
near
the
primary
veins
were
excised,
sectioned
into
small
pieces
and
frozen
in
liquid
nitrogen.
The
samples
were
then
freeze-dried
and
coated
with
gold
for
60
s
(ca.
1
nm
thickness
of
gold)
by
a
Sputter
Coater
(Cressington
model
108,
Ted
Pella
Inc.,
U.S.),
and
then
detected
by
a
scanning
electron
microscope
(SEM;
SU8010,
Hitachi,
Japan).
The
cross
sections
were
viewed
at
an
accelerating
potential
of
20
kV
under
high
vacuum
mode
with
backscatter
detection.
Images
were
captured
at
different
magnifications.
For
each
species,
at
least
three
plants
were
examined
for
each
treatment
group.
Digital
photographs
were
taken
using
an
EVO
40
scanning
electron
microscope
(Zeiss).
Methods
validation
We
used
two
fluorescent
markers
(Nile
blue
and
4-chloro-7-nitro-1,
2,
3-benzoxadiazole,
which
emitted
a
red
and
green
fluorescence
signal,
respectively,
Fig.
S4)
to
track
PS
beads
in
plant
tissues
and
found
fluorescence
to
be
a
sensitive
and
reliable
detection
method.
Plants
without
exposure
of
fluorescent
PS
beads
were
used
as
a
control
to
adjust
the
detector
gain
and
establish
the
baseline.
All
images
were
acquired
with
the
same
detector
gain
to
ensure
comparable
relative
intensities.
Under
the
imaging
conditions
used,
sections
from
untreated
control
plants
showed
no
detectable
autofluorescence
at
specific
excitation
(Fig.
S5).
When
roots
were
treated
with
fluorescent
PS
microbeads,
the
beads
could
be
identified
by
their
light
fluorescence.
The
roots
of
wheat
grown
in
0.2
m
m
microbead
suspension
showed
clear
concentration-dependent
fluorescence
in
a
wide
range
from
0.5–50
mg
L
–1
(Fig.
1),
thus
providing
evidence
for
increased
accumulation
of
beads
by
wheat
L.
Li
et
al.
/
MethodsX
7
(2020)
100750
3

plants
at
higher
treatment
concentrations.
Based
on
the
strong
fluorescence
signal
obtained
in
the
roots
of
wheat
when
grown
in
the
50.0
mg
L
–1
suspension
of
0.2
m
m
PS
beads,
50.0
mg
L
–1
suspensions
of
different
sizes
of
microbeads
(0.2.
2.0,
5.0.
or
7.0
m
m)
were
used
for
investigations
in
the
hydroponic
exposure
medium.
Significant
fluorescence
was
observed
in
the
roots,
shoots
and
leaves
of
wheat
exposed
to
0.2
m
m
beads
(Fig.
2).
Very
few
luminescence
signals
were
observed
in
the
vascular
system
or
in
the
epidermis
of
wheat
roots
for
2.0
m
m
and
almost
none
for
5.0
m
m
and
7.0
m
m
beads
(Fig.
3).
The
presence
of
aggregates
of
the
0.2
m
m
PS
beads,
mostly
in
the
xylem
and
on
the
cell
walls
of
the
cortex
tissue
in
the
wheat
root,
indicated
that
the
beads
passed
through
the
intercellular
space
via
the
apoplastic
transport
system
(Fig.
2).
Once
inside
the
central
cylinder,
particles
can
move
toward
the
aerial
parts
of
a
plant
though
the
xylem,
following
the
transpiration
stream.
The
0.2
m
m
PS
beads
were
transferred
from
the
roots
to
the
stems
and
leaves
via
the
vascular
system
through
the
apoplastic
Fig.
1.
Longitudinal
sections
of
the
mature
zone
of
roots
of
wheat
grown
in
Hoagland
solution
with
0.2
m
m
PS
microbeads
at
concentrations
of
0
(A–C),
0.5
(D–F),
5.0
(G–I)
or
50
(J–L)
mg
L
1
for
10
d.
PS
beads
were
labeled
with
Nile
blue.
The
accumulation
of
PS
beads
was
analyzed
under
bright-field
conditions
and
in
the
red
channel
using
CLSM.
Bar
=
100
m
m.
4
L.
Li
et
al.
/
MethodsX
7
(2020)
100750

pathway
to
the
leaf
vein
vasculature
(Fig.
2).
No
fluorescence
was
observed
in
the
stem
and
leaves
for
2.0
m
m
PS
beads.
Briefly,
our
observations
demonstrated
the
usefulness
of
using
fluorescent
labeled
MPs
for
studying
their
localization
within
the
whole
plant
tissues.
The
results
presented
here
clearly
demonstrate
the
uptake
and
transport
of
PS
microbeads
by
plant
roots
and
the
in
vivo
distribution
into
the
stem
and
leaves.
Our
work
reported
a
visualization
method
to
detect
the
accumulation
and
distribution
of
MPs
in
plant
tissues,
which
could
be
beneficial
to
profoundly
understand
the
biological
effects
and
the
modes
of
interaction
of
MPs
with
plants.
Additional
information
Microplastics
may
present
an
attributable
risk
to
ecosystem
and
human
health,
and
their
presence
in
the
biosphere
has
become
a
global
concern.
Terrestrial
edible
plants
are
for
instance
directly
exposed
to
MPs
when
organic
manure,
sewage
sludge
as
fertilizer,
or
plastic
mulching
[3–5]
are
applied
to
Fig.
2.
Confocal
images
of
cross
sections
of
a
wheat
root
(A–C),
stem
(D–F)
and
leaf
(G–I)
treated
for
10
d
with
a
50
mg
L
1
solution
of
0.2
m
m
fluorescently
labelled
polystyrene
(PS)
microbeads.
Images
A,
D,
and
G
are
the
corresponding
merged
images
of
image
B
and
C,
E
and
F,
H
and
I.
L.
Li
et
al.
/
MethodsX
7
(2020)
100750
5

agricultural
soil.
Despite
a
wealth
of
information
on
the
accumulation
of
MPs
in
aquatic
species
[6–8],
very
limited
information
on
the
uptake
and
accumulation
of
MPs
by
higher
plants
exists
to
date.
With
the
increasing
amounts
of
smaller
plastic
particles
directly
emitted
into
the
environment
as
well
as
secondary
particles
formed
by
degradation
of
plastics,
it
is
critical
to
understand
the
interactions
between
MPs
and
plants.
Studies
have
shown
the
possibility
of
plants
accumulating
small
size
microplastics
[9]
and
being
affected
either
positively
or
negatively
depending
on
plant
type
[10–14].
Information
regarding
the
accumulation
of
MP
by
plants
is
still
very
limited
and
the
underlying
mechanismof uptakeis
currently
not
clear.
Therefore,more
studies
are
requiredfor betterunderstanding
the
accumulation
and
distribution
of
MPs
in
the
plant.
For
intracellular
imagingof
MPs,
scanning
electron
microscopy
(SEM)
provides
high
resolution,showing
MP
shapes
and
sizesin
addition
totheirlocation
[9].
However,
identifying
MPs
using
SEM
or
TEM
requires
high
magnification,
resulting
in
a
limited
field
of
view.
In
addition,
the
extensive
sample
preparation
and
fixation
required
for
SEM
or
TEM
makes
it
Fig.
3.
Confocal
images
of
longitudinal
sections
in
the
root
zone
70
mm
from
the
root
apex
of
wheat
treated
for
10
d
with
a
50
mg
L
1
solution
of
2
m
m
(A–C),
5
m
m
(D–F)
or
7
m
m
(G–I)
polystyrene
(PS)
microbeads
labeled
with
Nile
blue.
The
accumulation
of
PS
beads
was
analyzed
in
the
red
channel
using
CLSM.
These
are
merged
bright-field
and
confocal
images.
Bar
=
100
m
m.
6
L.
Li
et
al.
/
MethodsX
7
(2020)
100750

challenging
to
explore
intracellular
trafficking
of
MPs
in
real
time.
Visualization
of
fluorescent
MPs
is
a
simple,
rapid,
and
non-invasive
approach
for
the
imaging
of
MPs
within
plant
tissues.
It
is
capable
of
rapidly
screening
whole
tissues
by
using
confocal
laser
scanning
microscope
(CLSM)
and
sample
preparations
are
simple.
No
additional
optical
or
imaging
system
is
required
for
this
approach.
Declaration
of
Competing
Interest
The
authors
report
no
conflicts
of
interest.
The
authors
alone
are
responsible
for
the
content
and
writing
of
the
paper.
Acknowledgements
This
work
is
supported
by
the
National
Key
Research
and
Development
Program
of
China
(Grant
No.
2016YFC1402202)
and
the
Key
Research
Project
of
Frontier
Science
(QYZDJ-SSW-DQC015)
of
the
Chinese
Academy
of
Sciences,
and
the
National
Natural
Science
Foundation
of
China
(41877142).
Appendix
A.
Supplementary
data
Supplementary
material
related
to
this
article
can
be
found,
in
the
online
version,
at
doi:https://doi.
org/10.1016/j.mex.2019.11.023.
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