Misin e p e a ion in mic oplas ic de ec ion in biological issues: When 2D
imaging is no enough
Alba Beni o-Kaesbach
a
, Jose Manuel Amigo
b,d
, U zi Izagi e
a
, Ne ea Ga cia-Velasco
a
,Lau a A é alo
c
,
And eas Sei e
b,c
, Kepa Cas o
d,
⁎
a
Cell Biology in En i onmen al Toxicology (CBET) Resea ch G oup, Dep . Zoology and Animal Cell Biology, Facul y o Science and Technology and Resea ch Cen e o Expe imen al Ma ine Biology and
Bio echnology PIE-UPV/EHU, Uni e si y o he Basque Coun y UPV/EHU, E-48080 Bilbao, Basque Coun y, Spain
b
IKERBASQUE, Basque Founda ion o Science, Euskadi Plaza 5, 48009 Bilbao, Spain
c
CIC nanoGUNE BRTA, Tolosa Hi ibidea 76, 20018 San Sebas ian, Spain
d
IBeA Resea ch G oup, Analy ical Chemis y Depa men , Facul y o Science and Technology, Uni e si y o he Basque Coun y UPV/EHU, E-48080 Bilbao, Basque Coun y, Spain
HIGHLIGHTS GRAPHICAL ABSTRACT
•We de ec ed 1 μm polys y ene pa icles by
Raman imaging in c yosec ions o mus-
sels.
•We de ec ed he mic oplas ics in he epi-
helium by 2D Raman imaging analysis.
•3D Raman imaging showed mic oplas ics
mo ed he e du ing sample p epa a ion.
•Spec oscopis s ha e o be awa e abou
possible imaging da a misin e p e a ion.
•Da a misin e p e a ion can also happen
when samples a e o ui ously con ami-
na ed.
ABSTRACTARTICLE INFO
Edi o : Damià Ba celó
Keywo ds:
Mic oplas ics
Raman imaging
Mussels
Polys y ene
C yo ome
The p esence o mic oplas ics in he ood chain is a public conce n wo ldwide, and i s analysis is an analy ical chal-
lenge. In ou esea ch, we apply Raman imaging o s udy he p esence o 1 μm polys y ene mic oplas ics in c yosec-
ions o My ilus gallop o incialis due o i s wide geog aphic dis ibu ion, widesp ead occu ence in he ood web, and
gene al high p esence in he en i onmen . Inges ed mic oplas ics a e accumula ed in he diges i e ac , bu a la ge
numbe can also be apidly elimina ed. Some au ho s s a e ha he ansloca ion o mic oplas ics o he epi helial
cells is possible, inc easing he isk o mic oplas ics ansmission along he ood chain. Howe e , as seen in ou
s udy, a su ace imaging app oach (2D) is p obably no enough o confi m he in e naliza ion o pa icles and a oid
misin e p e a ion. In ac , while some mic oplas ic pa icles we e de ec ed in he epi helium by 2D Raman imaging,
u he 3D Raman imaging analysis demons a ed ha hose pa icles we e d agged om he lumens o he epi helium
du ing sample p epa a ion due o he blade d ag e ec o he c yo ome, and subsequen ly loca ed on he su ace o he
analyzed c yosec ion, disca ding he ansloca ion o he epi helial cells. This e ec can also happen when he samples
a e o ui ously con amina ed du ing sample p epa a ion. Se e al esea ch a icles ha use simila analy ical ech-
niques ha e shown he p esence o mic oplas ics in di e en ypes o issue. I is no ou in en ion o pu such esul s
in doub , bu he p esen wo k poin s ou he necessi y o app op ia e h ee-dimensional analy ical me hods including
da a in e p e a ion and he need o go a s ep u he han jus su ace imaging analysis.
Science o he To al En i onmen 876 (2023) 162810
⁎Co esponding au ho .
E-mail add ess: kepa.cas [email protected] (K. Cas o).
h p://dx.doi.o g/10.1016/j.sci o en .2023.162810
Recei ed 24 Janua y 2023; Recei ed in e ised o m 6 Ma ch 2023; Accep ed 8 Ma ch 2023
A ailable online 13 Ma ch 2023
0048-9697/© 2023 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC license (h p://c ea i ecommons.o g/licenses/by-nc/4.0/).
Con en s lis s a ailable a ScienceDi ec
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1. In oduc ion
Rega dless o i s unlimi ed ange o applica ions and i s pi o al ole in
he de elopmen o mode n socie y, plas ic has become an en i onmen al
h ea . Fo example, big pa s o he p oduced polys y ene (PS), he 6 h
mos demanded plas ic in Eu ope in 2018 (De-la-To e e al., 2020), un o -
una ely ends up in he ma ine en i onmen (E ni-Cassola e al., 2017).
Public conce n o he po en ial ad e se e ec s o plas ics (Fang e al.,
2020) has been ocused on he so-called mic oplas ics (MPs), a g oup o
plas ic deb is in he ange o 1 μm o5mm(Jin-Feng e al., 2018;
Mendoza e al., 2018). Rega dless o how MPs a e gene a ed hey a e bio-
a ailable o a wide ange o in e eb a es (B owne e al., 2008), especially
o low ophic fil e eede s, de i i o es and plank i o ous (W igh e al.,
2013), because hey occupy he same size ac ion as sedimen s and plank-
onic o ganisms. The e o e, hey may accumula e wi hin o ganisms by di-
ec inges ion (Xu e al., 2019) o ophic ans e (Li e al., 2019),
p oducing physical ha m h ough in e nal ab asions and blockages.
Inges ed MPs a e accumula ed in hediges i e ac o mussels in a sho
ime. Howe e , a la ge numbe o hem can also be apidly elimina ed
(Calmão e al., 2023). Hence, i is impo an o de e mine he capaci y o
plas ics o be inco po a ed in o he cells o assess he consequen ial dam-
ages. I is p o en ha cells wi h phagocy ic ac i i y, such as mussel hemo-
cy e, can in e nalize MPs (Ka sumi i e al., 2021;Von Moos e al., 2012).
Ne e heless, cells used in hese s udies a e cul u ed cells ou o he p o ec-
ion o he lining epi helium and issue ba ie s. Thus, i needs mo e s udies
based on his ological and imaging app oaches o obse e he mic oplas ic
in e naliza ion in o cells and issues.
Sampling and analyses o MPs become mo e di ficul and demanding
he smalle he pa icles a e, which is one eason why mos o he MP su -
eys analyze only a size ange om 0.3 o 5 mm, a ange whe e isual
so ing is an app op ia e app oach (Lenz e al., 2015). Ne e heless, o gan-
isms such as fil e eede s cap u e and e ain 3–4μm pa icles wi h 100 %
e ficiency and can inges pa icles as small as 1 μm diame e (wi h a e-
duced e ficiency o 50 %) (W igh e al., 2013). Since small pa icles (<
100 μm) a e he mos ha m ul (P impke e al., 2020) and p esen ones in
he highes concen a ions (Ende s e al., 2015), mo e esea ch ega ding
hese sizes is equi ed.
To manage and con ol his eme ging pollu an , s anda dized me hods
o isola ion, de ec ion and iden ifica ion o MPs a e needed o pa icles
as small as 1 μm(No en a e al., 2021;Shim e al., 2017), which a e he a -
ge o his s udy.
Va ious me hods ha e been applied o isola e MPs in biological issues
(Yu e al., 2019), pa icula ly highligh ing he chemical diges ion me hod
(Zhu and Wang, 2020). Howe e , such echniques do no allow s udying
he spa ial dis ibu ion o MPs inges ed by he o ganism, which is key
om a oxicological poin o iew. I is impo an o know whe he hese
MPs accumula e in he mussel h ough inges ion a he han h ough adhe -
ence o he su ace o he issue (Kolandhasamy e al., 2018).
In con as , c yosec ion echnique is e y use ul o in es iga ing he
ansloca ion o MPs o o he issues and could se e o in es iga ing he
exac posi ion o he pa icles inges ed by li ing o ganisms (Colla d e al.,
2017). I azola e al. (I azola e al., 2015) used c yosec ion and pa a fin-
embedded sec ions o s udy he accumula ion and dis ibu ion o MPs in
di e en issues o mussels My ilus gallop o incialis, concluding ha ozen
issue allows o be e de ec ion o MPs han pa a fin-embedded issue.
In ac , MPs we e dissol ed du ing he sample p epa a ion in pa a fin, as o -
ganic sol en s a e usually used.
Rega ding he de ec ion and iden ifica ion s ep, he e a e many analy -
ical ools (Xu e al., 2019). Howe e , he mos common echniques used o
analyze MPs in biological samples a e Fou ie - ans o m in a ed (FTIR)
and Raman spec oscopy (Käpple e al., 2016), which equi e small sample
amoun s, a e applicable wi h high accu acy, and allow e en o he de e -
mina ion o pa icle size dis ibu ions (A aujo e al., 2018;Ele e al.,
2017).
As he diame e o he lase beam is smalle in Raman spec oscopy, his
me hod allows o he iden ifica ion o pa icles o smalle size down o
1μm(A aujo e al., 2018;Nguyen e al., 2019;Xu e al., 2019). Howe e ,
sca ce s udies ha e epo ed he iden ifica ion o 1 μm MPs, mainly because
hei iden ifica ion depends also on o he pa ame e s such as he complex-
i y o he sample (di ficul isola ion), applied fil e ype, and measu emen
pa ame e s (Nguyen e al., 2019;Xu e al., 2019), among o he s.
Despi e he ypical Raman poin analysis, which has been ex en-
si elyapplied os udyMPsinbiological samples o ma ine o ganisms
(Dehau e al., 2016;Li e al., 2019), chemical imaging o his pu pose
has begun qui e ecen ly. Fo example, Leung e al. (2021) used an au o-
ma ed mapping app oach o de ec 5 di e en ypes o plas ics in
diges ed samples o Pe na i idis, de ec ing pa icles in a size ange o
30–820 μm.
Chemical imaging echniques a e accep ed as p omising ools o MPs
analysis in su aces (Fang e al., 2020;Xu e al., 2019), pa icula ly when
s uc u ed samples, such as di ec analysis o issues, a e unde s udy.
The e o e, when a biological ma ix (such as c yosec ion) is mapped by
Raman spec oscopy, i migh be possible o specifically iden i y whe e
he MPs a e accumula ed inside he mussel, o de e mine he ela i e
amoun , and e en he size o he pa icles. Howe e , as Ele e al., 2017 in-
es iga ed, he spec oscopic scanning me hods a e clea ly mo e ime-
consuming han he ex ac ion me hods. Fo example, Imho e al., 2016
and Vianello e al., 2013 epo ed ha only ≈2%o ≈5 % o he sample
could be analyzed in Raman imaging, espec i ely.
Ne e heless, one o he ad an ages o spec oscopic echniques, as o
example Raman imaging, is he analysis o pa icle size dis ibu ion by i-
sual image, a ea u e ha can only be s udied wi h high- esolu ion mic os-
copy echniques (Ele e al., 2017). In compa ison o Raman imaging, FTIR
imaging leads o significan unde es ima ion (abou 35 %) o MPs, espe-
cially in he size ange lowe han 20 μm. Un o una ely, he measu emen
ime o Raman imaging is qui e long compa ed o FTIR imaging (Käpple
e al., 2016).
In a nu shell, one me hod alone will no ulfil all he equi emen s o
p o ide a comp ehensi e da ase o wide- anging de ec ion o MPs.
Raman imaging yields mo e de ailed in o ma ion abou he chemis y o
he pa icles and hei size and dis ibu ion. Howe e , i p esen s disad an-
ages such as he limi ed sample su ace ha can be in es iga ed in a single
un in a easonable ime and equen di ficul ies wi h con amina ion o he
o ganic ma ix in he sample, which could be conside ed as impo an
d awbacks o applying his me hod o as e alua ion o MPs in he
en i onmen .
In gene al, chemical imaging analysis (such as Raman o FTIR imaging
analysis) has been unde s ood as he iden ifica ion o specific molecules on
a su ace (2D imaging analysis). Tha way, mic oplas ics analysis has been
ca ied ou , oo, as in Le e mo e e al., 2020. As p esen ed in his esea ch
wo k, he cu en echnical capabili ies o mode n ins umen a ion allow
o olume ic chemical analysis (3D imaging analysis) hanks o he high
con ocali y o mode n mic oscopes (Ma son e al., 2013), which opens
up new in e es ing analy ical app oaches o s udying mic oplas ics
(Ta a da e al., 2022). In e es ing esea ch a icles employing 3D Raman
imaging o analyze cells can be ound in li e a u e (Kallepi is e al., 2017;
Lin e al., 2020;Wei e al., 2019). Howe e , only a ew s udies use his ech-
nique o mic oplas ics analysis, o example, Chen e al., 2017, who s udy
polys y ene beads o 10 and 100 μm diame e in 3D olumes and lipid d op-
le s in adipose cells.
In ou esea ch, we s udy he capabili y o Raman imaging as a use ul
echnique o s udy he p esence and dis ibu ion o 1 μm polys y ene MPs
in c yosec ions o biological ma ices.By i ue o i s wide geog aphic dis-
ibu ion, sessile li es yle, basal posi ion on he ood web, easy sampling, i s
well-unde s ood biology, and high accumula ion o a wide ange o pollu -
an s (including MPs), M. gallop o incialis was chosen as he model o ganism
in ou s udy. Mo eo e , i ep esen s an animal o human consump ion,
which adds alue o i s use in his ype o s udy. The p oposed app oach
iden ifies he dis ibu ion o pa icles inside he o ganism, pa icula ly in
he diges i e gland as he a ge o gan in his wo k. We will poin ou pos-
sible da a misin e p e a ion based on 2D (su ace) imaging, and highligh
he benefi s o 3D ( olume) imaging.
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
2
2. Expe imen al sec ion
2.1. Mic oplas ic s anda ds and mussel samples
As polys y ene is one o he mos used plas ics, one o he mos p esen
in ma ine en i onmen s, and i deg ades qui e easily and gene a es
mic oplas ics o a high ex en , comme cial fluo escen mic osphe es o
polys y ene (PS) wi h a size o 1 μm we e used o ca y ou he expe imen
(Fluo o-Max™G een Fluo escen Polyme Mic osphe es, The mo Fishe
Scien ific, ca alogue numbe G0100). The MP solu ion had a concen a ion
o 1 % (w/w) o fluo escen mic osphe es and a densi y o 1.05 g/cm
3
.To
a oid misin e p e a ion due o c oss-con amina ion o he biological issues
wi h mic oplas ics om he en i onmen (labo a o y, clo hes, wa e , e c.),
we used labeled pa icles, fluo escen pa icles in his case.
My ilus gallop o incialis (4.82 ±0.8 cm -maximumdis ance be ween he
umbo and he pos e io ma gin o he shell; 1.56 ± 0.5 g-biomass, mean ±
SD) we e collec ed om he es ua y o Bu on i e , in Plen zia, Bizkaia,
43°24′33.6”N 2°56′51.5”W, on he 14 h o Ma ch 2021. Mussels we e col-
lec ed om he ocks when he ide was low by ca e ully cu ing byssal
h eads o a oid damaging he unde lying so issues. 15 mussels we e col-
lec ed, which we e subsequen ly washed wi h seawa e o emo e he e-
mains o sand and o ganic ma e . Once cleaned, hey we e le in a ank
ull o seawa e wi h cons an wa e enewal o ca y ou hei depu a ion
in he Plen zia Ma ine S a ion, Uni e si y o he Basque Coun y (PiE-
UPV/EHU) o a leas 48 h o allow elimina ion o he gu con en s be o e
analysis o he so issues (So o e al., 1995).
2.2. Se up o sample p epa a ion
Exposu es o PS MPs we e pe o med in ood absence, pe sis en ae a-
ion, a cons an empe a u e o 16 °C and a 12/12 h ligh /da k egime.
Two glass beake s (300 mL) we e filled wi h 250 mL o seawa e p e iously
fil e edwi h a 0.2 μmfil e . In each glass beake 3 mussels we e in oduced,
one beake se ing as con ol, he o he o exposu e o PS wi h 5.5 × 10
6
pa icles/mL.
We used an MP concen a ion much highe han hose eco ded in ma-
ine en i onmen s o ensu e ha he mussels e ain plas ics. The highe he
concen a ion, he mo e ce ain one can be o find he pa icles. Applicable
doses in labo a o y es s ha e been discussed (Lenz e al., 2016)and
c i icised in esea ch wo ks ocused on up ake a ios, MP o al mass calcu-
la ions in animals, epidemiologic/incidence/ oxicological s udies, e c.
Howe e , his is no he pu pose o ou esea ch wo k he e. The conclusions
o ou s udy do no ocus on he amoun o mic oplas ics p esen in he is-
sues, o on he a e o ansloca ion in o he issues, bu a he on whe he
o no hey a e inside he issue, and whe he ou analy ical app oach al-
lows us o de ec hem including spa ial in o ma ion.
The mussels we e exposed o he pa icles o 24 h, which is long
enough o hem o fil e MPs in he wa e column. Mussels we e main-
ained wi h cons an in ense ae a ion o maximize buoyancy and ensu e
ha he MPs s ayed in he wa e column o as long as possible. The mussels
we ecollec ed a e he exposu e o MPs, and hei shells we e washed wi h
dis illed wa e o p e en con amina ion and emo e MPs adso bed on he
shell su aces. La e , he shell was emo ed, and he inne so issue was
ca e ully isola ed and p epa ed o c yosec ion. All glasswa e and ins u-
men s we e washed wi h dis illed wa e o p e en con amina ion.
The loca ion o he MPs in mussel issueswas s udied using c yosec ion-
ing. A c oss-sec ion o he so issue was ozen wi h liquid ni ogen. Fo
his pu pose, he sample was placed on a floa ing me allic pla e o ensu e
slow eezing and a oid c ys alliza ion. The sample was posi ioned such
ha a comple e c oss-sec ion o he mussel could be p epa ed. Once he
so issues we e ozen, hey we e s o ed in a me allic pla e in a eeze
a −40 °C un il use. Tissue sec ions we e p epa ed wi h a Leica CM
3050S C yos a equipped wi h a mo o ized cu ing de ice a a chambe
empe a u e o −25 °C. The c oss-sec ions o he mussels we e fixed o a
me allic chuck by adding a c yo-embedding medium. The c yosec ions o
10 μm hickness we e collec ed on o glass slides and kep a oom
empe a u e, causing issue sec ions o flash-d y immedia ely upon con ac .
Two o h ee issue sec ions we e collec ed on o e e y glass slide. All he
c yosec ions we e quickly sc eened unde he Leica Mic oscope. Sec ions
wi h he bes condi ions and he g ea es p opo ion o he diges i e
gland we e chosen o sea ch he MPs and o s udy he dis ibu ion o he
pa icles in he lumen and epi helium o he diges i e ac .
Du ing sec ioning, all blades and an i- olls we e changed a e each
ea men , and all ma e ials in di ec con ac wi h he samples we e cleaned
wi h ace one o a oid c oss-con amina ion by MPs. Blanks we e used o e -
i y ha he cleaning o he c yo ome was e ec i e.The blankswe e mussels
ee o fluo escen MPs (no ed wi h fluo escen MPs). C yosec ions o
hese mussels we e analyzed unde a fluo escence mic oscope o e i y
ha no MPs we e p esen , ha is, no MP con amina ion was p oduced in
he c yo ome.
2.3. Se up o Raman spec oscopy and imaging
A Renishaw inVia con ocal Raman mic oscope (Renishaw, Glouces e -
shi e, UK), was used o Raman poin -by-poin and imaging analysis. The
measu emen s we e ca ied ou wi h a 532 nm exci a ion lase (Renishaw,
Glouces e shi e, UK, RL532C50) wi h a nominal ou -pu powe o 300 mW.
The sys em has a CCD de ec o cooled by Pel ie e ec (−70 °C), and i is
coupled o a Leica DMLM mic oscope (B ad o d, UK) ha allows he mic o-
scopic analysis o he sample by using 5× N PLAN (0.12 NA), N PLAN EPI
(0.40 NA), and 50× N PLAN (0.75 NA) and 100× (LMPlanFL N 0.80 NA)
objec i es. The nominal powe o he sou ce goes om 0.05 % o 100 % o
he o al powe . To a oid he mal decomposi ion o he sample, he nomi-
nal powe o he lase was adjus ed.
Fo Raman image mapping, he samples we e scanned using a 50× ob-
jec i e lens ( heo e ical lase spo size diame e on he ocal plane a
532 nm is 0.87 μm), wi h an in eg a ion ime o 3.5–5 s and a spa ial eso-
lu ion o 1 μm/pixel in all axes (XYZ), a an ope a ing spec al esolu ion
smalle han 1 cm
−1
. To gene a e he image, he ne in ensi y o he main
cha ac e is ic peak o polys y ene MPs a 1001 cm
−1
(see supplemen a y
ma e ial Fig. S1) was plo ed in he mapped a ea. Baseline co ec ion,
smoo hing and cosmic ay emo al o he acqui ed spec a we e pe o med
wi h he Renishaw WiRE 4.2 so wa e, as well as o ob ain he Raman im-
ages. The InVia spec ome e was calib a ed daily, se ing he 520.5 cm
−1
silicon line.
2.4. Se up o fluo escence mic oscopy
In addi ion, a fluo escence pho omic oscope (Olympus BX50) was used
o confi m he p esence o he fluo escen PS MPs in he analyzed c yosec-
ions o he mussels. The mic oscope is equipped wi h op ical fil e s o isu-
alize di e en wa eleng hs. In his wo k, 488 nm was used as exci a ion
wa eleng h, and he fluo escen g een line a 515 nm was de ec ed. O igin
6.0 so wa e was used o da a analysis and plo ing he spec a o he MPs.
As i is echnically almos impossible o find mic opa icles in la ge mac o-
scopic a eas in easonable ime by con ocal Raman imaging (see below),
ull-field imaging wi h fluo escence mic oscopy was ca ied ou be o e-
hand, which allows us o easily iden i y suspicious pa icles and a eas on
which one can ocus by Raman spec oscopy hen.
3. Resul s and discussion
3.1. Mic oplas ics in biological samples: Raman imaging
To ad ance sample p epa a ion o op imized mic oscopy imaging e-
ga ding he iden ifica ion o MPs, sec ions o 10 μm hickness we e ana-
lyzed. In a fi s s ep, fluo escen PS MPs we e de ec ed by molecula
fluo escence o ensu e ha MPs we e inside he diges i e sys em o he
mussels. Then, he mos p omising a eas o he c oss-sec ions o he mussels
we e selec ed o u he Raman analysis. Poin -by-poin Raman analyses
we e ca ied ou in bo h he lumen and he epi helium o he s omach
and gu .
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
3
The band o he a oma ic ca bon ing ha appea s a 1001 cm
−1
was
used o confi m he p esence o PS in he sample (Fig. 1). The small size
o he pa icles, oge he wi h he signal coming om he issue o he mus-
sels (sample ma ix, see Fig. 1b), makes he de ec ion o MPs challenging. In
addi ion o PS MPs, o he pa icles inside he diges i e sys em we e ound,
which u ned he finding o MPs in o a ial-and-e o app oach.
As can be seen, he numbe o PS MPs in he lumen (Fig. 1c) was highe
han in he epi helium(Fig. 1a). In ac , agg ega ion o pa icles was no iced
in he lumen. In he Raman spec a ob ained om he PS MPs loca ed in he
epi helium, wo s ong and wide bands due o he ma ix we e always e-
co ded a a ound 550 and 1100 cm
−1
. Howe e , in all cases, he ea u es
o PS MPs we e isible (see he s anda d spec um o fluo escen PS MPs
in Fig. S1).
Once being ce ain ha he MPs could be obse ed by poin Raman
analysis, Raman imaging wasapplied o de ec he MPs in a mo e au oma ic
and less ial-and-e o manne . Fo ha , a smallsec ion o he s omach was
mapped using mo o ized s ages wi h s eps o 1 μm o bo h he X and Y axis.
Tha way, a spec um wi h 1 μm pe pixel was ob ained (see Fig. 2 as an ex-
ample).
The scanned a ea is indica ed by he ec angula egion in Fig. 2 and
co esponds o a su ace o 387 μm × 151 μm. The ime o mapping his
a ea wi h s eps o 1 μm×1μm was 64 h. In Fig. 2 we can see, in ed, he
PS MPs de ec ed by Raman imaging a e moni o ing he in ensi y a
1001 cm
−1
o e he isible image o he mussel ma ix. The colo channels
o he isible image in he figu e ha e been modified o be e con as and
isualiza ion o he MPs.
As seen in Fig. 2, a high amoun o PS MPs was accumula ed in he
lumen o he diges i e ac , dis ibu ed all along he longi udinal mapped
sec ion o he s omach. This fi s Raman image was used o asce ain he
abili y o Raman o de ec MPs au oma ically and o explo e and fix he op-
imal pa ame e s o u he analysis in he epi helium in he same condi-
ions. To he bes o ou knowledge, his is he fi s ime ha pa icles as
small as 1 μm a e de ec ed in biological samples using Raman imaging. Al-
hough a sec ion o he epi helium was mapped, no pa icles we e obse ed
in ha specific a ea. Thus, he majo i y o he MPs obse ed in mussels
we e in he luminal ca i y o diges i e sys ems, which is in acco dance
wi h he obse a ions by Calmão e al., 2023.
Cells wi h phagocy ic ac i i y, such as mussel hemocy e, can in e nalize
MPs (Ka sumi i e al., 2021;Von Moos e al., 2012). Howe e , he capaci y
o MPs o c oss e y well-o ganized epi heliums, such as he diges i e ac
epi helium, and ge inco po a ed in o issues, isno en i ely p o en. Fo he
basic biological unc ions o any o ganism,i isessen ial o keep he luminal
(ou e ) side, and he issue (inne ) side sepa a ed. P esen ly, he unc ion o
hese ba ie s appea ed o be p ope ly main ained since he as majo i y o
Fig. 1. Fluo escen polys y ene MPs in c yosec ions o My ilus gallop o incialis ma ked wi h ed ci cles. a) In gu epi helium a 50× magnifica ion and co esponding Raman
spec um in b). c) In gu lumen a 50× magnifica ion wi h co esponding Raman spec um in d). (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade
is e e ed o he web e sion o his a icle.)
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
4
MPs obse ed in he o ganisms we e in he lumen and no in e nalized in
epi heliums and cells. Damage o he cell memb ane could condi ion he en-
ance o MPs in o he epi helium. Ne e heless, li le is known abou he
eal memb ane damage ha would allow MPs o en e he epi helial cells
o be ween hem. Recen ly, appa en cell damage ela ed o MPs was ob-
se ed (Wang e al., 2021). Howe e , unde e y di e en expe imen al se -
ings wi h ex emely high MP doses in cell cul u e. I he biological ba ie s
we e egula ly su passed by MPs o in animals unde mul iple en i onmen-
al s ess, he accumula ion in issuesand ans e o highe le el o ganisms
in he ophic ne would be exace ba ed. To ully unde s and his p oblem,
i is o u mos impo ance o de elop powe ul ools o he iden ifica ion o
plas ics inside issues. The app oach used in he p esen s udy could illumi-
na e eal in e naliza ion in o he cells and issues o MPs and de e mine
hei eal influence and di ec damage.
To decide whe he Raman imaging is use ul o mapping and de ec ing
pa icles inside he epi helium, fi s , he c yosec ions we e obse ed unde
he fluo escence mic oscope (Fig. 3a, colo channels o he image ha e been
modified o be e con as and isualiza ion). The c yosec ion wi h a
highe con en o MPs in ha a ea was chosen o ca y ou a Raman map-
ping (Fig. 3b and c), whose scanning a ea is ma ked wi h a ec angle and
co esponds o a su ace o 21 μm×38μm. The ime needed o map his
a ea was 55 min wi h an in eg a ion ime o 4 s, yielding a e y good
signal- o-noise a io om he MPs. The Raman image (Fig. 3c) was gene -
a ed ollowing he same s eps as he ones in Fig. 2.InFig. 4, wo ypical
Raman spec a a e ep esen ed. The one in ed colo is he a e aged spec-
um o he a ea whe e PS MPs we e ound (showing he main Raman
band a 1001 cm
−1
), whe eas he one in black is he a e aged spec um
o he a ea whe e he mussel issue was measu ed wi hou MPs.
Acco ding o he esul s shown in Fig. 3, one migh hink ha he e a e
some PSMPs inside he epi helium o he gu ; hus, ou wo k ep esen s one
o he fi s s udies in which he ansloca ion o MPs o he issue has been
p o ed by Raman imaging a e MPs up ake by mussels.
Howe e , due o he ubiqui ous occu ence o MPs nowadays, o ui ous
con amina ion o he sample due o MPs deposi ion ac oss he su ace o he
c yosec ion is a pe manen h ea , e en unde well-con olled labo a o y
condi ions, as o example in clean ooms, labo a o y and equipmen
cleaning, labwa e and lab clo hes con ol. Mo eo e , e en hough he c yo-
s a mic o ome uses a sha p blade o cu he issue samples in o hin slices,
MPsmigh bemo ed by heblade (blade d ag e ec ) om diges i e lumens
(eg. s omach, gu ) oo he issues, a ac ha would in alida e he p oposed
conclusions due o da a misin e p e a ion. To ou knowledge, his con o-
e sial issue has no been discussed in any p e ious wo k.
Mic oplas ics in biological samples: Volume ic Raman chemical imag-
ing (VRI).
To p o e whe he he pa icles we e ac ually ansloca ed om he
lumen o he gu o he epi helium and in e nalized by epi helial cells, a
ull h ee-dimensional (3D) mapping (Volume ic Raman chemical imag-
ing) om he gu epi helium was ca ied ou . This map gi es in o ma ion
abou he composi ion, posi ion, size and mo phology o he sample,
allowing us o know he loca ion o he pa icles in all h ee spa ial dimen-
sions (X, Y and Z).
In Fig. 5, di e en adjacen c oss-sec ional planes o he epi helium a e
shown, sepa a ed by 1 μm dep h. A o al o 10 planes a e shown. The num-
be 0 co esponds o he su ace o he c yosec ion, and he o he planesa e
he ones unde nea h he su ace o he c yosec ion. This map has a su ace
o 84 μm×69μm, mul iplied by 10 planes, so ha a o al o 72 h we e nec-
essa y o acqui e he en i e 3D spec al cuboid (1μm×1μm×1μmmo o
s ep o he mic oscope s age). The Raman image was gene a ed by he in-
ensi y o he cha ac e is ic Raman peak a 1001 cm
−1
o PS MPs (in
g een colo in Fig. 5) and by he in ensi y o he Raman band a
1008 cm
−1
, cha ac e is ic o he ma ix o he mussel (in ed colo in
Fig. 5).
The image shows ha he g een do s only appea ed in he fi s planes.
F om he plane loca ed a −4μm on he Z axis, he signal o he MPs disap-
pea ed, and only signals om he mussel ma ix we e obse ed. These ob-
se a ions demons a e ha he pa icles we e no in e nalized by
epi helial cells o be ween epi helial cells, since i hey we e, he pa icles
would ha e appea ed a di e en dep hs o he epi helium and would be
su ounded by he epi helium. No MPs su ounded by issue we e ound,
which pu s in doub ou ini ial esul s and conclusions ega ding he ac ual
p esence o MPs in he epi helium.
Some au ho s suppo he hypo hesis ha he ansloca ion o MPs o
he epi helial cells could be done by phagocy osis, endocy osis, o ano he
mechanism ha allows he pa icles o pass h ough he in es inal ba ie
(Colla d e al., 2017). Du ing his p ocess, a g ea e numbe o small MP
pa icles can appea in he epi helium because he smalle pa icles a e
Fig. 2. Visible image (mosaic) o he analyzed s omach sec ion ob ained unde a 50× objec i e o a DMLM Leica mic oscope coupled o he inVia Raman spec ome e . The
mapped sec ion o he s omach in which he fluo escen PS MPs o 1 μm we e ound is highligh ed in he da k ec angle. The ed poin s a e he fluo escen PS MPs de ec ed
hanks o he in ensi y o i s Raman band a 1001 cm
−1
. (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his
a icle.)
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
5
mo e easily phagocy osed, possibly because he phagosomes wi hin each
cell can accommoda e smalle pa icles (B owne e al., 2008).
Howe e , as seen in ou s udy, a su ace imaging app oach migh no be
enough o confi m he in e naliza ion o pa icles and a oid misin e p e a-
ion. C yosec ioning has been an es ablished echnique o many yea s,
pa icula ly applied o me hods ha do no suppo adi ional his ological
echniques o when immedia e esul s a e equi ed (Nguyen e al., 2019).
Ne e heless, ou p esen wo k e eals ha e en i he sample p epa a ion
seems co ec , ca e mus be aken when analyzing he esul s om Raman
o any o he high- esolu ion imaging echnique o a oid misin e p e a ion.
In any case, some s udiesha e highligh ed he p esence o plas ic mic o-
pa icles in he s omach epi helium (B owne e al., 2008).Fo example,Von
Moos e al., 2012 de e mined he p esence o MPs 0–80 μm in diges i e ep-
i helial cells, sugges ing ha pa icles we e aken up ia he mou h,
anspo ed o he gas oin es inal ac , and in e nalizedin ocells o he di-
ges i e sys em by endocy osis. González-So o e al., 2019 obse ed indi id-
ual 4.5 μm MPs wi hin epi helial cells o he diges i e ac , duc s, and
diges i e ubules. Colla d e al., 2017 epo ed he p esence o la ge
(124 μm - 438 μm) MPs in he li e o he Eu opean ancho y. None o
hese wo ks, as many o he s in he li e a u e, discuss possible misin e p e-
a ion de i ed om he sample p epa a ion and he c yo ome/mic o ome
blade d ag e ec on MPs, ha is, MPs d agged om he lumens o he epi-
helium du ing sample p epa a ion.
In Raman spec oscopy, he esolu ion o an image is cons ained by
he di ac ion limi o he exci a ion lase spo (Fang e al., 2020). Due o
he di ac ion limi , he smalles la e al in o ma ion olume is he Ai y
disc, which is a unc ion o he nume ical ape u e and he exci a ion wa e-
leng h. Ano he limi ing ac o can be he pixel size o he came a/de ec o .
Recen ly, Raman imaging was used o iden i y nanoplas ics o 100 nm
(Sobhani e al., 2020). Ne e heless, when mapped, he pa icles in he
image showed a la ge size o 300 nm –400 nm, which is p obably a esul
o he di ac ion limi o he lase spo when ocused on he nanopa icles
o scanning and emi ing he Raman signal (Fang e al., 2020). Unde
hese conside a ions, i can be explained why in Fig. 5 he PS mic opa icles
a e ep esen ed much la ge han hei eal size, when mapping hei
Raman signal. The di ac ion limi is e en mo e un a o able when he
pa icle size dec eases, making analysis o e y small pa icles almos
impossible. Ano he possible explana ion would be ha he obse ed mi-
c opa icles a e no indi idual pa icles bu agg ega ions o se e al ones.
4. Conclusions
Due o hei small size and in e e ence wi h o ganicand en i onmen al
ma e ial, sepa a ing and iden i ying mic oplas ic pa icles in biological
samples is challenging. Howe e , we we e able o e ficien ly cha ac e ize
polys y ene mic opa icles down o a size o 1 μm in c yosec ions om
Fig. 3. a) Fluo escence mic oscopy image o PS MPs, showing in ed he a ea wi h highe amoun o PS mic obeads in he gu epi helium. b) Visible image (mosaic) o he
whole s omach sec ion ob ained unde a 50× objec i e o a DMLM Leica mic oscope coupled o he inVia Raman spec ome e ; in ed he mapped a ea o Raman
mic oscopy. c) Raman image o he mapped sec ion o he epi helium in which he de ec ed fluo escen PS mic oplas ics o 1 μm a e shown in ed. (Fo in e p e a ion o
he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
6
Fig. 4. A e aged Raman spec a o mussel issue. Red: a ea on issue wi h PS MPs wi h i s cha ac e is ic peaka 1001 cm
−1
; black: issue wi hou plas ics. (Fo in e p e a ion
o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
Fig. 5. Rep esen a ion o he Z planes o he a ea o he gu epi helium mapped. Each image ep esen s a di e en dep h, wi h 1 μm di e ence in hickness be ween adjacen
planes. G een colo ep esen s he fluo escen PS MPs, and he ed colo ep esen s he mussel ma ix. (Fo in e p e a ion o he e e ences o colo in his figu e legend, he
eade is e e ed o he web e sion o his a icle.)
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
7
My ilus gallop o incialis by Raman imaging. Sobhani e al., 2020 demon-
s a ed ha Raman mapping was use ul o image and isualize an indi id-
ual nanoplas ic pa icle wi h a diame e o 100 nm, di ec ly placed on a
gold-coa ed mic oscope slide. Gi en ha i has been demons a ed ha
MPs o 1 μm size can be iden ified in biological samples using Raman imag-
ing, nex impo an esea ch s eps ha e o ocus on he de ec ion and iden-
ifica ion o nano and molecula plas ics, which p obably play a much
s onge ad e se ole in biological sys ems.
Some pa icles o 1 μm size we e obse ed in he gu epi helium, which
could be one o he fi s de ec ions o such small MPs in animal issue. How-
e e , a u he olume ic Raman chemical imaging analysis showed ha
he mic oplas ics we e loca ed on he su ace o he analyzed c yosec ion.
We poin ou he mic o ome blade d ag e ec as a possible eason o he
p esence o mic oplas ics on he su ace o he analyzed epi helium. E en
hough c yosec ion is s ill a highly use ul p epa a ion echnique ha allows
o isualiza ion o pa icles di ec ly on mussel issue whe e hey a e na u-
ally accumula ed, and gi es mo e biologically ele an in o ma ion han
o he mo e commonly used me hods, as o example chemical diges ion,
p ecau ions ha e o be aken o a conclusi e da a in e p e a ion.
In li e a u e, esea ch can be ound ha shows mic oplas ic p esence in
se e al issues by analyzing hem wi h analy ical echniques simila o hose
used in he p esen wo k. I is no ou in en ion o pu such esul s in doub ,
bu he p esen wo k poin s ou he necessi y o app op ia e da a in e p e a-
ion and he need o go a s ep u he han jus su ace imaging analysis. As a
esul , i u ns ou ha olume ic Raman imaging analysis is an impo an
and essen ial analy ical echnique o analyze he dis ibu ion o pa icles
and confi m hei p esence a issue and cellula le els; o he wise, alsepos-
i i e de ec ions and w ong conclusions could easily be d awn.
Based on ou analy ical findings, we conclude ha su ace mapping is
insu ficien o cha ac e izing and loca ing mic oplas ics in and on issue
slices, as olume ic Raman imaging analysis shows ha he mic opa icles
did no ansloca e in o epi helial cells, a leas , no in ou analyzed sam-
ples. In o he wo ds, su ace mapping is su ficien o iden i ying MPs, bu
gi es limi ed in o ma ion abou he eal 3D loca ion in o on issue.
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.sci o en .2023.162810.
CRediT au ho ship con ibu ion s a emen
Alba Beni o-Kaesbach: Pape w i ing, Raman imaging measu emen s.
Jose Manuel Amigo: Da a ea men (imaging), Figu e edi ing.
U zi Izagi e: Pape w i ing, Sampling o he mussels and all expe i-
men s ela ed wi h he mussels eeding wi h mic oplas ics.
Ne ea Ga cia-Velasco: Sampling o he mussels and all expe imen s e-
la ed wi h he mussels eeding wi h mic oplas ics.
Lau a A é alo: Da a ea men (imaging).
And eas Sei e : Pape w i ing and co ec ing, coo dina o o he expe -
imen s and s a .
Kepa Cas o: Pape w i ing, Raman imaging measu emen s,
Figu e edi ing.
Da a a ailabili y
Da a will be made a ailable on eques .
Decla a ion o compe ing in e es
The au ho s decla e he ollowing financial in e es s/pe sonal ela ion-
ships which may be conside ed as po en ial compe ing in e es s: Kepa Cas-
o epo s financial suppo was p o ided by Basque Business
De elopmen Agency. And eas Sei e epo s financial suppo was p o-
ided by Minis y o Science Technology and Inno a ions. And eas Sei e
epo s financial suppo was p o ided by Basque Business De elopmen
Agency. U zi Izagi e epo s financial suppo was p o ided by Minis y
o Economic A ai s and Digi al T ans o ma ion. U zi Izagi e epo s fi-
nancial suppo was p o ided by Basque Business De elopmen Agency.
Acknowledgemen s
This wo k was unded by Basque Go e nmen (KK 2021/00001
ELKARTEK 2021/2022, IT1743-22); MINECO (PID 2020-118685RB-I00,
PLASTeMER); u he financial suppo by g an CEX2020-001038-M
unded by MCIN/AEI/ 10.13039/501100011033.
Re e ences
A aujo, C.F., Nolasco, M.M., Ribei o, A.M., Ribei o-Cla o, P.J., 2018. Iden ifica ion o
mic oplas ics using Raman spec oscopy: la es de elopmen s and u u e p ospec s.
Wa e Res. 142, 426–440.
B owne, M.A., Dissanayake, A., Galloway, T.S., Lowe, D.M., Thompson, R.C., 2008. Inges ed
mic oscopic plas ic ansloca es o he ci cula o y sys em o he mussel, My ilus edulis
(L.). En i on. Sci. Technol. 42, 5026–5031.
Calmão, M., Blasco, N., Beni o, A., Thoppil, R., To e-Fe nandez, I., Cas o, K., Izagi e, U.,
Ga cia-Velasco, N., So o, M., 2023. Time-cou se dis ibu ion o fluo escen mic oplas ics
in a ge issues o mussels and polychae es. Chemosphe e 311, 137087.
Chen, X., Zhang, C., Lin, P., Huang, K.C., Liang, J., Tian, J., Cheng, J.X., 2017. Volume ic
chemical imaging by s imula ed aman p ojec ion mic oscopy and omog aphy. Na .
Commun. 8, 15117.
Colla d, F., Gilbe , B., Compè e, P., Eppe, G., Das, K., Jauniaux, T., Pa men ie , E., 2017.
Mic oplas ics in li e s o Eu opean ancho ies (Eng aulis enc asicolus, L.). En i on. Pollu .
229, 1000–1005.
Dehau , A., Cassone, A.L., F è e, L., He mabessie e, L., Himbe , C., Rinne , E., Ri iè e, G.,
Lambe , C., Soudan , Ph., Hu e , A., Duflos, G., Paul-Pon , I., 2016. Mic oplas ics in sea-
ood: benchma k p o ocol o hei ex ac ion and cha ac e iza ion. En i on. Pollu . 215,
223–233.
De-la-To e, G.E., Dioses-Salinas, D.C., Piza o-O ega, C.I., Saldaña-Se ano, M., 2020. Global
dis ibu ion o wo polys y ene-de i ed con aminan s in he ma ine en i onmen : a e-
iew. Ma . Pollu . Bull. 161, 111729.
Ele , A.M., Becke , R., Duemichen, E., Eisen au , P., Falkenhagen, J., S u m, H., B aun, U.,
2017. Compa ison o di e en me hods o MP de ec ion: wha can we lea n om
hem, and why asking he igh ques ion be o e measu emen s ma e s? En i on. Pollu .
231, 1256–1264.
Ende s, K., Lenz, R., S edmon, C.A., Nielsen, T.G., 2015. Abundance, size and polyme compo-
si ion o ma ine mic oplas ics≥10 μm in he A lan ic Ocean and hei modelled e ical
dis ibu ion. Ma . Pollu . Bull. 100, 70–81.
E ni-Cassola, G., Gibson, M.I., Thompson, R.C., Ch is ie-Oleza, J.A., 2017. Los , bu
ound wi h Nile ed: a no el me hod o de ec ing and quan i ying small
mic oplas ics (1 mm o 20 μm) in en i onmen al samples. En i on. Sci. Technol.
51, 13641–13648.
Fang, C., Sobhani, Z., Zhang, X., Gibson, C.T., Tang, Y., Naidu, R., 2020. Iden ifica ion and i-
sualisa ion o mic oplas ics/nanoplas ics by aman imaging (ii): smalle han he di ac-
ion limi o lase ? Wa e Res. 183, 116046.
González-So o, N., Ha field, J., Ka sumi i, A., Du oudie , N., Laca e, J.M., Bilbao, E., O bea,
A., Na a o, E., Caja a ille, M.P., 2019. Impac s o die a y exposu e o di e en sized
polys y ene mic oplas ics alone and wi h so bed benzo[a]py ene on bioma ke s and
whole o ganism esponses in mussels My ilus gallop o incialis. Sci. To al En i on. 684,
548–566.
Imho , H.K., La o sch, C., Wiesheu, A.C., Schmid, J., Ange , P.M., Niessne , R., I le a, N.P.,
2016. Pigmen s and plas ic in limne ic ecosys ems: a quali a i e and quan i a i e s udy
on mic opa icles o di e en size classes. Wa e Res. 98, 64–74.
I azola, M., Ga mendia, L., Zaldiba , B., Izagi e, U., Bilbao, E., Danielsson, S., Bigne , A.,
Cas o, K., E xeba ia, N., So o, M., Ma igomez, I., 2015. Combina ion o mic oscopic
and spec oscopic echniques o s udy he p esence and he e ec s o mic oplas ics in
mussels. P oceedings o he MOL2NET, Sci o um Elec onic Con e ence Se ies h ps://
doi.o g/10.3390/MOL2NET-1-c010.
Jin-Feng, D.I.N.G., Jing-Xi, L.I., Cheng-Jun, S.U.N., Chang-Fei, H.E., Jiang, F.H., Feng-Lei,
G.A.O., Zheng, L., 2018. Sepa a ion and iden ifica ion o mic oplas ics in diges i e sys em
o bi al es. Chin. J. Anal. Chem. 46, 690–697.
Kallepi is, C., Be ghol , M.S., Mazo, M.M., Leona do, V., Skaalu e, S.C., Mayna d, S.A.,
S e ens, M.M., 2017. Quan i a i e olume ic Raman imaging o h ee dimensional cell
cul u es. Na . Commun. 8, 14843.
Käpple , A., Fische , D., Obe beckmann, S., Sche newski, G., Lab enz, M., Eichho n, K.J., Voi ,
B., 2016. Analysis o en i onmen al mic oplas ics by ib a ional mic ospec oscopy:
FTIR, Raman o bo h? Anal. Bioanal. Chem. 408, 8377–8391.
Ka sumi i, A., Losada-Ca illo, M.P., Ba os, M., Caja a ille, M.P., 2021. Polys y ene
nanoplas ics and mic oplas ics can ac as ojan ho se ca ie s o benzo(a)py ene o mus-
sel hemocy es in i o. Sci. Rep. 11, 22396.
Kolandhasamy, P., Su, L., Li, J., Qu, X., Jabeen, K., Shi, H., 2018. Adhe ence o mic oplas ics
o so issue o mussels: a no el way o up ake mic oplas ics beyond inges ion. Sci. To al
En i on. 610, 635–640.
Lenz, R., Ende s, K., S edmon, C.A., Mackenzie, D.M., Nielsen, T.G., 2015. A c i ical assess-
men o isual iden ifica ion o ma ine mic oplas ic using Raman spec oscopy o analy-
sis imp o emen . Ma . Pollu . Bull. 100, 82–91.
Lenz, R., Ende s, K., Nielsen, T.G., 2016. Mic oplas ic exposu e s udies should be en i onmen-
ally ealis ic. PNAS 113, E4121–E4122.
Leung, M.M.L., Ho, Y.W., Lee, C.H., Wang, Y., Hu, M., Kwok, K.W.H., Chua, S.L., Fang, J.K.H.,
2021. Imp o ed Raman spec oscopy-based app oach o assess mic oplas ics in sea ood.
En i on. Pollu . 117648.
Le e mo e, J.M., Smi h, T.E., Kelly, F.J., W igh , S.L., 2020. De ec ion o mic oplas ics in am-
bien pa icula e ma e using aman spec al imaging and chemome ic analysis. Anal.
Chem. 92, 8732–8740.
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
8
Li, J., Lushe , A.L., Ro chell, J.M., Deude o, S., Tu a, A., B å e, I.L.N., Sun, C., Hossain, M.S.,
Li, Q., Kolandhasamy, P., Shi, H., 2019. Using mussel as a global bioindica o o coas al
mic oplas ic pollu ion. En i on. Pollu . 244, 522–533.
Lin, P., Ni, H., Li, H., Vicke s, N.A., Tan, Y., Gong, R., Bi ano, T., Cheng, J.X., 2020. Volume ic
chemical imaging in i o by a emo e- ocusing s imula ed Raman sca e ing mic oscope.
Op . Exp ess 28, 30210–30221.
Ma son, E.C., Aboualizadeh, E., Ba abas, M.E., S ucky, C.L., Hi schmugl, C.J., 2013. Oppo u-
ni ies o li e cell FT-in a ed imaging: mac omolecule iden ifica ion wi h 2D and 3D lo-
caliza ion. In . J. Mol. Sci. 14, 22753–22781.
Mendoza, L.M.R., Ka apanagio i, H., Ál a ez, N.R., 2018. Mic o (nanoplas ics) in he ma ine
en i onmen : cu en knowledge and gaps. Cu . Opin. En i on. Sci. Heal h 1, 47–51.
Nguyen, B., Cla eau-Malle , D., He nandez, L.M., Xu, E.G., Fa ne , J.M., Tu enkji, N., 2019.
Sepa a ion and analysis o mic oplas ics and nanoplas ics in complex en i onmen al sam-
ples. Accoun s Chem. Res. 52, 858–866.
No en a, S., Boyles, M.S.P., Sei e , A., Belluco, S., Jiménez, A.S., Johns on, H.J., T an, L.,
Fe nandes, T.F., O sini, M., Co ami, F., Cas o, K., Mu inelli, F., Bold in, M., Pun es, V.,
So oudeh, M., Masca ello, G., Tiozzo, B., McLean, P., Ronchi, F., Boo h, A.M.,
Koelmans, A.A., Losasso, C., Mughini-G as, L., 2021. Pa adigms o assess he human
heal h isks o nano- and mic oplas ics. Mic oplas ics and Nanoplas ics 1 (9).
P impke, S., Godejohann, M., Ge d s, G., 2020. Rapid iden ifica ion and quan ifica ion o
mic oplas ics in he en i onmen by quan um cascade lase -based hype spec al in a ed
chemical imaging. En i on. Sci. Technol. 54, 15893–15903.
Shim, W.J., Hong, S.H., Eo, S.E., 2017. Iden ifica ion me hods in mic oplas ic analysis: a e-
iew. Anal. Me hods 9, 1384–1391.
Sobhani, Z., Zhang, X., Gibson, C., Naidu, R., Megha aj, M., Fang, C., 2020. Iden ifica ion and
isualisa ion o mic oplas ics/nanoplas ics by Raman imaging (i): down o 100 nm.
Wa e Res. 174, 115658.
So o, M., Ko abi a e, M., Ma igómez, I., 1995. Bioa ailable hea y me als in es ua ine wa e s
as assessed by me al/shell-weigh indices in sen inel mussels My ilus gallop o incialis.
Ma . Ecol. P og. Se . 125, 127–136.
Ta a da , A., Choi, S.H., Kwon, J.H., 2022. Di e en ial s aining lowe s he alse posi i e de ec-
ion in a no el olume ic measu emen echnique o mic oplas ics. J. Haza d. Ma e .
432, 128755.
Vianello, A., Bold in, A., Gue ie o, P., Moschino, V., Rella, R., S u a o, A., Da Ros, L., 2013.
Mic oplas ic pa icles in sedimen s o lagoon o Venice, I aly: fi s obse a ions on occu -
ence, spa ial pa e ns and iden ifica ion. Es ua . Coas . Shel Sci. 130, 54–61.
Von Moos, N., Bu kha d -Holm, P., Köhle , A., 2012. Up ake and e ec s o mic oplas ics on
cells and issue o he blue mussel My ilus edulis L. A e an expe imen al exposu e. En-
i on. Sci. Technol. 46, 11327–11335.
Wang, S., Hu, M., Zheng, J., Huang, W., Shang, Y., Fang, J.K.H., Shi, H., Wang, Y., 2021. In-
ges ion o nano/mic o plas ic pa icles by he mussel My ilus co uscus is size dependen .
Chemosphe e 263, 127957.
Wei, M., Shi, L., Shen, Y., Zhao, Z., Guzman, A., Kau man, L.J., Wei, L., Min, W., 2019. Volu-
me ic chemical imaging by clea ing-enhanced s imula ed aman sca e ing mic oscopy.
PNAS 116, 6608–6617.
W igh , S.L., Thompson, R.C., Galloway, T.S., 2013. The physical impac s o mic oplas ics on
ma ine o ganisms: a e iew. En i on. Pollu . 178, 483–492.
Xu, J.L., Thomas, K.V., Luo, Z., Gowen, A.A., 2019. FTIR and Raman imaging o mic oplas ics
analysis: s a e o he a , challenges and p ospec s. T AC T ends Anal. Chem. 119,
115629.
Yu, J., Wang, P., Ni, F., Cizdziel, J., Wu, D., Zhao, Q., Zhou, Y.Ma , 2019. Cha ac e iza ion o
mic oplas ics in en i onmen by he mal g a ime ic analysis coupled wi h Fou ie ans-
o m in a ed spec oscopy. Pollu . Bull. 145, 153–160.
Zhu, J., Wang, C., 2020. Recen ad ances in he analysis me hodologies o mic oplas ics in
aqua ic o ganisms: cu en knowledge and esea ch challenges. Anal. Me hods 12,
2944–2957.
A. Beni o-Kaesbach e al. Science o he To al En i onmen 876 (2023) 162810
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