Ad ancing mic oplas ic de ec ion in zeb a ish wi h mic o compu ed
omog aphy: A no el app oach o e ealing mic oplas ic dis ibu ion
in o ganisms
Vik ´
o ia Pa obko ´
a
a
, Luk´
aˇ
s Maleˇ
cek
a
, Ma ek Zemek
a
, Gab iela Kalˇ
cíko ´
a
b,c,*
,
Michaela Vykypˇ
elo ´
a
, Ma cela Buch o ´
a
b,d,e
, Ondˇ
ej Adamo ský
, Tom´
aˇ
s Zikmund
a,b,*
,
Joze Kaise
a,b
a
Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyˇ
no a 656/123, B no 61200, Czech Republic
b
Facul y o Mechanical Enginee ing, B no Uni e si y o Technology, Technick´
a 2896/2, B no 61669, Czech Republic
c
Facul y o Chemis y and Chemical Technology, Uni e si y o Ljubljana, Veˇ
cna po 113, Ljubljana 1000, Slo enia
d
Depa men o Expe imen al Biology, Facul y o Science, Masa yk Uni e si y, B no 625 00, Czech Republic
e
Labo a o y o Molecula Mo phogenesis, Ins i u e o Animal Physiology and Gene ics, Czech Academy o Sciences, B no 602 00, Czech Republic
RECETOX, Facul y o Science, Masa yk Uni e si y, Ko l´
aˇ
sk´
a 2, B no 611 37, Czech Republic
HIGHLIGHTS GRAPHICAL ABSTRACT
•The no el me hodology o de ec ion o
mic oplas ics in zeb a ish.
•Mic oCT enabled 3D, non-in asi e
isualiza ion o mic oplas ics.
•Spa ial dis ibu ion o mic oplas ics in
he gas oin es inal ac was obse ed.
•The p oposed me hodology de ec ed
polye hylene mic oplas ics as small as
30 µm.
ARTICLE INFO
Keywo ds:
Mic oplas ics
X- ay Compu ed Tomog aphy
En i onmen
Plas ic Pollu ion
Imaging
ABSTRACT
The analysis o mic oplas ics wi h cu en spec oscopic and py oly ic me hods is eaching i s limi s, especially
wi h ega d o de ailed spa ial dis ibu ion in biological issues. This limi a ion hampe s a comp ehensi e un-
de s anding o he e ec s o mic oplas ics on o ganisms. The e o e, he e is a p essing need o expand he
analy ical app oaches o s udy mic oplas ics in bio a. In his con ex , he aim o his s udy was o es he
applicabili y o non-des uc i e 3D imaging using X- ay mic o-compu ed omog aphy (mic oCT) o he de ec-
ion o mic oplas ics in ish. Zeb a ish (Danio e io) we e ga aged wi h polye hylene sphe ical mic oplas ics
(30–110
μ
m) and he dis ibu ion o mic oplas ics in he gu was in es iga ed using mic oCT. The esul s showed
* Co esponding au ho s a : Facul y o Mechanical Enginee ing, B no Uni e si y o Technology, Technick´
a 2896/2, B no 61669, Czech Republic.
E-mail add esses: [email p o ec ed] (G. Kalˇ
cíko ´
a), [email p o ec ed] (T. Zikmund).
Con en s lis s a ailable a ScienceDi ec
Jou nal o Haza dous Ma e ials
jou nal homepage: www.else ie .com/loca e/jhazma
h ps://doi.o g/10.1016/j.jhazma .2025.137442
Recei ed 21 No embe 2024; Recei ed in e ised o m 10 Janua y 2025; Accep ed 28 Janua y 2025
Jou nal o Haza dous Ma e ials 488 (2025) 137442
A ailable online 28 Janua y 2025
0304-3894/© 2025 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license ( h p://c ea i ecommons.o g/licenses/by/4.0/ ).
ha he pa icle size dis ibu ion de e mined by mic oCT closely ma ched he da a om con en ional lase
di ac ion analysis. In addi ion, mic oCT was able o de ec mic oplas ics in spiked ish issue and p o ide
p ecise localiza ion da a by acing pa icles o known ype and shape. Mic oCT o e s a no el app oach o
acking mic oplas ics in o ganisms and enables accu a e sizing wi hou comp omising he in eg i y o he issue
unde in es iga ion. I he e o e ep esen s a aluable addi ion o spec oscopic me hods, which a e widely used
o he de ec ion o mic oplas ics based on hei chemical composi ion bu do no p o ide da a on hei spa ial
dis ibu ion.
1. In oduc ion
The plas ic pollu ion is cu en ly conside ed a wo ldwide p oblem
due o i s widesp ead dis ibu ion and pe sis ence in he en i onmen . In
addi ion o he mac oplas ic pollu ion, mic oplas ics (MPs, plas ic
agmen s om 1 o 1000
μ
m [1]) ha e become a se ious issue due o
hei nega i e impac s on he en i onmen [2,3] and possibly also
human heal h [4–6].
O e he las decade, se e al s udies ha e been conduc ed on how
MPs in e ac wi h animal issues and a ec a ious o ganisms [7]. The
esul s indica e ha hey can en e he o ganism p ima ily h ough
inhala ion and/o inges ion [8]. In biological samples polye hylene (PE)
is he mos equen ly de ec ed MP, ollowed by polyp opylene (PP),
polys y ene (PS), and polye hylene e eph hala e (PET) [9–11]. MPs can
be ound in a ious shapes, including agmen s, ib es, beads, and ilms
[10]. The p esence o MPs in o ganisms can lead o se e e nega i e e -
ec s - s uc u al damage o he in es ine, li e , and bo h he exc e o y
and ep oduc i e sys ems, oxida i e s ess, al e a ions in
immune- ela ed gene exp ession, and modi ica ions in an ioxidan s a-
us [4,12–19]. Mo eo e , la es esea ch indica es ha unde s anding
he dis ibu ion o MPs in he o ganism is c ucial o he e alua ion o
hei heal h e ec s and accumula ion in o gans [6,20,21].
The e o e, o link he e ec s o MPs on he o ganism, he e idence o
MPs dis ibu ion in speci ic issues is essen ial. Se e al new app oaches
o a eliable MPs de ec ion in biological issues ha e been de eloped
la ely. Con en ionally, wo ypes o de ec ion me hods a e used; py ol-
ysis and spec oscopy [22]. In case o py oly ic me hods, he sample is
hea ed and he MPs a e deg aded in o simple o ganic compounds [23].
The ola ile py olysis p oduc s a e sepa a ed by gas ch oma og aphy
and de ec ed by mass spec ome y. In his app oach, he esul s a e
gi en as a mass o polyme de ec ed in he issue, bu he sizes and
numbe o MPs canno be e alua ed. Mo eo e , s udies e ealed ha
py oly ic me hods can lead o alse de ec ion o MPs in issue due o
in e e ence caused by molecules like a y acid- ype lipids, wi hin he
issue, in he quan i ica ion o MPs [24]. Spec oscopic me hods, e.g.,
Raman spec oscopy and Fou ie ans o m in a ed spec oscopy
(FTIR), can eliably de ec speci ic pa icles so he in o ma ion on hei
sizes and numbe is gi en. Howe e , hese me hods ace signi ican
challenges, wi h he main d awback being he sample p epa a ion e-
qui emen s, such as diges ion o sec ioning, which ca y a high isk o
pa icle loss and con amina ion. These issues can lead o misleading
esul s [25–27]. Consequen ly, he local dis ibu ion o pa icles wi hin
he analysed issue canno be de e mined due o a des uc i e sample
p epa a ion, p ecluding he assessmen o a heal h isk posed by he
accumula ion o MPs in di e en issue egions [28]. Mo eo e , he issue
o a seconda y con amina ion du ing sample p epa a ion emains un-
esol ed, as mos published s udies lack p ope con ols o assess he
c oss-con amina ion [29].
This s udy p esen s he i s explo a ion o mic oCT as a me hod o
de ec ing MPs in zeb a ish. Mic oCT o e s non-des uc i e imaging wi h
a high spa ial esolu ion, enabling he 3D analysis o MP dis ibu ion and
mig a ion wi hin a selec ed o ganism. While his me hod p o ides many
bene i s, analysing MPs in biological samples using mic oCT has no
been widely applied o in oduced in p ac ice ye . Only a ew s udies
u ilized mic oCT o MP de ec ion, p ima ily ocusing on MPs in sedi-
men s [30–32]. In con as o con en ional MP de ec ion me hods,
mic oCT does no equi e des uc i e sample p epa a ion, allowing o a
di ec iden i ica ion o MPs inside issues wi hou he isk o seconda y
con amina ion o loss o he MP’s spa ial dis ibu ion.
Howe e , challenges may a ise while using his me hod, namely he
di icul y in de ec ing pa icles ha a e nea o smalle han he oxel
size, making hem po en ially unde ec able in he olume ic da a. The
size o de ec able pa icles is closely ied o he oxel size, whe e oxel
size close o ew mic ome e (o e en submic on whe e easible) allow
o he isualiza ion o ine de ails, including smalle pa icles. How-
e e , ine oxel size also necessi a es a smalle ield o iew, equi ing
sample o be o limi ed dimensions. The e o e, ade-o be ween he
esolu ion and sample size is necessa y. To add ess his, we employed
wo di e en CT se ups in his s udy o wo sample ypes – whole
zeb a ish and hei ex ac ed gu s. The i s se up o e ed a la ge ield o
iew, sui able o scanning o he en i e abdominal egion in zeb a ish
samples. The second se up, op imized o scanning o he ex ac ed
zeb a ish gu s, p o ided a highe esolu ion by educing he ield o
iew, allowing o a po en ially lowe minimum de ec able pa icle size.
To explo e he po en ial o mic oCT, MPs made o PE we e selec ed o
he op imiza ion, as PE is a polyme p oduced in a high quan i y and PE
MPs a e o en ound in he en i onmen [11].
In gene al, he low a enua ion o polyme ic ma e ials o X- ays
needs o be add essed as i may lead o a educed image con as .
The e o e, we assessed whe he iodine s aining enhanced o hinde ed
MP de ec ion. To in es iga e his, we es ed he di ec de ec ion o PE
MPs in s ained biological samples using he op imized zeb a ish and gu s
se ups. Consequen ly, his s udy highligh s he po en ial o mic oCT o
de ec ing MPs in zeb a ish samples and lays he ounda ion o u he
esea ch o op imize his echnique o human samples.
2. Ma e ial and me hods
2.1. Mic oplas ics
MPs used in his s udy we e sphe ical PE pa icles wi h a size ange
om 45
μ
m o 75
μ
m pu chased om Cosphe ic LLC (USA). Thei size
was also e alua ed by a pa icle size analyse (S3500 Bluewa e lase
di ac ion analyse , Mic o ac, Ge many). MPs we e placed in he d y
uni , and he measu emen was epea ed h ee imes, e ealing ha he
ac ual size dis ibu ion o he pu chased ba ch anged om 30
μ
m o 110
μ
m. The p ima y eason o selec ing sphe ical pa icles was ha hei
uni o m shape and size enable a eliable compa ison o measu emen s
ob ained wi h a con en ional lase di ac ion analyse and mic oCT.
2.2. P epa a ion o e e ence samples
Ini ially, he mic oCT measu emen s o MP de ec ion using wo
di e en mic oCT se ups was op imized. MP concen a ion o 0.7 % (w/
), p o iding a e y dense abundance o MPs, was p epa ed in aga ose
gel which was chosen o i s simila i y o so issue in X- ay a enua ion
aspec s [33], acili a ing quick and p ac ical easibili y e i ica ion
(Fig. 1 A), and scanned based on he equi emen s o wo se ups used
u he : Zeb a ish (Fig. 1 B) and Gu s Se ups (Fig. 1 C) (se ups a e
u he explained in Sec ion 2.4).
V. Pa obko ´
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Jou nal o Haza dous Ma e ials 488 (2025) 137442
2
2.3. P epa a ion o model samples
In o de o con i m he applicabili y o he me hodology o complex
biological samples, we used he adul zeb a ish (Danio e io) as model
o ganisms and he ga age app oach. Zeb a ish a e among he mos
widely used model o ganisms in biology and eco oxicology, o e ing an
excellen pla o m o a ious s udies. They a e equen ly employed o
in es iga e he e ec s o MPs on o ganisms [34]. Using mic oCT in such
esea ch could p o ide aluable insigh s in o how MPs mig a e wi hin
he zeb a ish diges i e sys em, whe he hey accumula e, and, i so, he
speci ic loca ions o accumula ion.
In agas ic ga age was pe o med on wo adul zeb a ish. Ini ially,
an amoun o 2 mg o PE MPs in 100 µL e hanol was gi en o he ish
gas oin es inal ac by ul a ine pipe e ips. Speci ically, o ensu e
smoo h passage o he mic oplas ics, he ip o he pipe e was sligh ly
cu o enla ge he hole. The zeb a ish we e eu hanized in a icaine so-
lu ion (0.3 mg/mL) o ensu e p ecise deli e y o he solu ion. The
pipe e ip was ca e ully inse ed in o he esophagus, a oiding inju y o
he ish. The solu ion was dispensed slowly o ensu e he mic osphe es
en e ed he gas oin es inal ac . No e, ha o op imize and ensu e he
easibili y o de eloping a no el mic oCT me hodology o de ec ing
MPs and e alua ing hei dis ibu ion in he gu , we in en ionally used a
concen a ion highe han en i onmen ally ele an le els. Then, he
wo ish we e eu hanized by o e dosing by T icaine Me hanesul ona e
(MS222, Me ck). Two ish we e dissec ed, and he whole dissec ed in-
es ine was placed in o a his ological casse e, he o he wo ish we e
p ocessed in ac . All samples (whole ish o hei dissec ed gu s) we e
placed in 4 % o maldehyde in PBS and s o ed in a idge be o e u he
p ocessing. This esea ch, using zeb a ish, was app o ed by he E hical
Commi ee o he Czech Minis y o Educa ion, You h and Spo s unde
app o al numbe MSMT-12088/2024–4.
P io o he mic oCT measu emen , whole ish and ex ac ed gu s
we e s ained in 1 % iodine (17570–30250, Pen a) in 90 % me hanol
(21210–11000, Pen a) o 12 hou s a 4◦C. To elimina e issue sh ink-
ing, he samples we e passed h ough an e hanol dehyd a ion se ies -
30 %, 50 %, 70 %, 80 % and 90 % - each o 2 hou s. Subsequen ly,
based on he p epa a ion o he e e ence samples (Fig. 1 B, C), zeb a ish
we e placed in o a 1.5 ml Eppendo ube while he ex ac ed gu s we e
se in o a 3 mm diame e Kap on ube o he mic oCT measu emen .
Bo h ubes we e hen illed wi h 1 % aga ose gel (P045, Top-BIO) o
ensu e s abili y du ing acquisi ion.
2.4. Mic oCT measu emen s o mic oplas ics
Two CT se ups we e op imized o di e en sample ypes. Fi s , he
Zeb a ish Se up was de eloped o isualize he en i e gas oin es inal
ac (GIT) o p epa ed zeb a ish, allowing o an accu a e analysis o
MPs mig a ion and accumula ion wi hin he in ac sample. Second, he
Gu s Se up wi h a educed ield o iew was designed o analyse
ex ac ed gu s. This se up is also sui able o a u u e use in examining
small samples o dissec ed o gans wi h a a he high de ail. The da a
we e acqui ed using he Nano3DX de eloped by Rigaku, Japan. This
de ice is equipped wi h a high-powe X- ay sou ce (1200 W) wi h a
o a ing anode. Scanning pa ame e s o bo h se ups a e lis ed in
Table 1. Consequen ly, a il e ed back p ojec ion was used o econs uc
he acqui ed p ojec ions. To mi iga e de iciencies, s anda d algo i hms
o ing a i ac educ ion and de e mina ion o he cen e o o a ion
shi we e applied du ing he econs uc ion p ocess.
2.5. Segmen a ion o mic oplas ics
The econs uc ed da a we e p ocessed and analysed using a VG
S udio Max 2023.4 so wa e (Volume G aphics GmbH, Ge many) (Fig. 2
A). The same segmen a ion wo k low was used o bo h e e ence and
model samples. The pa icle segmen a ion was conduc ed using he
Pain and Segmen module, which u ilizes a neu al ne wo k o seg-
men a ion. Two label g oups we e c ea ed: 1. PE MPs and 2. Back-
g ound. A eas om se e al slices we e manually segmen ed using a
Fig. 1. P epa a ion o e e ence samples: (A) PE MPs we e imme sed in aga ose gel, (B) ans e ed in o a 1.5 ml PCR ube, chosen o i wi hin he ield o iew o
he Zeb a ish Se up, enabling a oxel size o 4.1 µm. (C) Addi ionally, he mix u e was placed in o a 3 mm diame e Kap on ube, i ing he ield o iew wi hin he
Gu s Se up. The highligh ed ields o iew we e hen scanned using mic oCT.
Table 1
Scanning pa ame e s o CT se ups. Values anno a ed wi h as e isk we e modi-
ied o e e ence samples.
Se up Zeb a ish Gu s
Voxel Size [µm] 4.2 2.1
Field o iew [mm] 7.13 ×5.4 3.56 ×2.7
Lens L2160 L1080
Ta ge Coppe *
Molybdenum
Coppe *
Molybdenum
Vol age [keV] 40 *
50
40 *
50
Cu en [mA] 30 *
24
30 *
24
Fil e 0.1 mm Aluminium il e 0.1 mm Aluminium il e
Exposu e ime [ms] 9 *
5
7 *
5
Numbe o P ojec ions 800 800
Binning 2 2
V. Pa obko ´
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Jou nal o Haza dous Ma e ials 488 (2025) 137442
3
pain b ush ool o iden i y PE MPs and anno a e he emaining a eas as a
backg ound. An au oma ic segmen a ion was hen e ined by inco po-
a ing hese manually segmen ed egions in o he classi ica ion p ocess
(Fig. 2 B).
Mo eo e , he gu s we e manually segmen ed in model samples o
enhance he isualiza ion o he spa ial dis ibu ion o e ie ed MPs in
3D ende ings and o p e en he de ec ion o MPs ou side he gu e-
gion. By in e sec ing he manually segmen ed gu egion wi h he
de ec ed pa icles, MPs loca ed ou side he gu we e excluded as
ou lie s.
Nex , he diame e o he segmen ed pa icles was de e mined by
employing he Po osi y/Inclusion module (Fig. 2 C).
The ’Only h eshold’ algo i hm was execu ed wi h a p e-segmen ed
egion om he Pain and Segmen module. Wi hin he se ings, he
de ia ion was modi ied o ensu e ha he whole egion o segmen ed
pa icles is o e lapped in p e iew o his algo i hm o ensu e a alid
diame e calcula ion o all he de ec ed pa icles om he p e-segmen ed
da a. Addi ionally, ’Inclusion’ was se as he indica ion ype and a
p obabili y h eshold was se be ween 1 and 1.5 o elimina e alse de-
ec ions, such as noise anno a ed as pa icles in Pain and Segmen
module.
E en ually, he size dis ibu ion o he p ocessed esul s was plo ed
using MATLAB (R2022b, Ma hWo ks), while he segmen a ion esul s,
along wi h he size anno a ions o he segmen ed pa icles, we e isu-
alized h ough a 3D ende in VG S udio (Fig. 2 D). This isualiza ion
o e ed a clea pe spec i e o he analysed gu olume.
2.6. Quali y assessmen o he mic oCT da a
The in ensi y his og ams we e ex ac ed using VG S udio o es ima e
he con as be ween he su ounding a ea and PE MPs. Fo e e ence
samples, he su ounding a ea’s in ensi ies we e ob ained om he
cen al egion o he da ase , while o model samples, hey we e de i ed
om wi hin he gu s, excluding egions con aining PE MPs in bo h cases.
A second his og am was gene a ed o ep esen he in ensi y dis ibu ion
o he pa icles in he same egions used o he su ounding a ea cal-
cula ions. The peak in ensi ies om bo h his og ams we e hen iden i-
ied, and he di e ences be ween hese peak alues we e calcula ed o
es ima e he con as be ween he aga ose gel o su ounding issue and
he pa icles o bo h expe imen al se ups. These egions we e u he
used o subsequen calcula ions.
The Con as - o-Noise Ra io (CNR) assessed he con as be ween PE
MPs and he backg ound, no malized by he noise le el in he back-
g ound. The CNR was calcula ed using he o mula [35]:
CNR =|xPE MPs −xBCG|
µBCG
Fig. 2. The p ocess o segmen a ion and analysis o MPs. Fo isualisa ion pu poses, he model zeb a ish sample acqui ed using he Zeb a ish Se up was u ilized. The
low included: (A) da a acquisi ion and ende ing, (B) MP segmen a ion, (C) diame e quan i ica ion, and (D) 3D ende ing o segmen ed pa icles colo -labeled based
on he de e mined diame e .
V. Pa obko ´
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Jou nal o Haza dous Ma e ials 488 (2025) 137442
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Whe e xPE MPs and xBCG a e he mean g ay alues wi hin PE MPs and
backg ound egions. µBCG s ands o he s anda d de ia ion o he
backg ound which de ines he su ounding egions. Ra he high CNR
alues indica ed a good isibili y o he PE MPs agains he backg ound.
2.7. S a is ical analyses
S a is ical analyses we e u ilized o assess he simila i y be ween he
samples and he esul s om pa icle size analyse . Gaussian dis ibu-
ions we e i ed o he da a om each sample, which we e i s
no malized o a ange o 0–1 and hen in e pola ed o ensu e a smoo h
ep esen a ion. A wo-sample - es was used o compa e he means o
each sample agains he mean o he pa icle size dis ibu ion, wi h he
null hypo hesis s a ing ha he e was no signi ican di e ence be ween
he means. An F- es was employed o compa e he a iances o each
sample agains he a iance o he pa icle size dis ibu ion, wi h he null
hypo hesis asse ing ha he e was no signi ican di e ence be ween he
a iances. Bo h es s we e conduc ed using a signi icance le el o
α
=0.05 and we e pe o med using MATLAB.
2.8. Mo phological analysis o pa icles
The pe cen age o MP abundance nea he in es inal illi was
calcula ed by di iding he numbe o MPs loca ed close o he illi by he
o al numbe o segmen ed pa icles. A mask o he in e nal gu egion
was i s c ea ed o classi y MPs based on hei p oximi y o he illi o
he cen e o he gu . This mask was hen mo phologically e oded,
meaning we sh ank i o o m a mo e compac shape, oughly 100 µm
away om he illi endings. MPs wi hin his e oded egion we e ca e-
go ized as "close o he cen e," while he emaining pa icles ou side he
e oded mask we e conside ed "close o he illi."
The abundance o MPs in di e en egions o he in es ines was
quan i ied by di iding he segmen ed gu in o h ee dis inc sec ions:
an e io , middle, and pos e io . The egion be o e he i s u n was
designa ed as he an e io , he sec ion ollowing he las u n as he
pos e io , wi h he middle egion loca ed in be ween. These egions
we e manually segmen ed, and he numbe o PE MPs wi hin each
sec ion was de e mined by ex ac ing pa icles om he o iginal seg-
men a ion o each egion indi idually.
To u he assess he MP dis ibu ion along he leng h o he colon, a
polyline was d awn in VG S udio. This ool allowed us o measu e each
indi idual MP’s dis ance om he gu ’s beginning. We d ew a polyline
om each pa icle’s posi ion o he gu ’s s a . Addi ionally, o calcula e
he o al leng h o he gu , we used he polyline ool o ace a line om
he beginning o he end o he gu .
3. Resul s
3.1. P elimina y isualiza ion o mic oplas ics
Wi h a ocus only on e e ence samples, PE MPs anging om 30 o
110 µm we e embedded in aga ose gel and isualized using mic oCT.
Two di e en scanning se ups we e es ed (Zeb a ish and Gu s), a ying
in scanning pa ame e s and equi ed sample dimensions. The la ge
sample, gene a ed in a PCR ube and scanned wi h he Zeb a ish Se up
yielded a oxel size o 4.2 µm (Fig. 3A, A‘), while he smalle sample,
p epa ed in a 3 mm diame e Kap on ube and measu ed using he Gu s
Se up, esul ed in a oxel size o 2.1 µm (Fig. 3B, B‘).
Since mic oCT isualizes ma e ials based on hei densi ies, he PE
MPs appea ed as da ke oxels in he da ase due o hei lowe densi y
compa ed o he aga ose gel. This con as enabled he de ec ion and
segmen a ion o MPs in ob ained da a.
Nex , he MP segmen a ion was success ully comple ed using au o-
ma ic ools. Howe e , some MPs we e no included in he segmen a ion
(Fig. 4A, B). By analysing selec ed 0.6 mm³ a eas wi hin he scanned
da a and compa ing manual and au oma ed segmen a ion, he esul s
indica ed ha he Zeb a ish Se up had an a e age o 22.5 % missed
pa icles ou o he o al pa icles coun ed, all o which we e below
40 µm in size. Al hough, using he same compa ison app oach o he
Gu s se up, 100 % o pa icles we e de ec ed.
Fig. 3. Mic oCT da a acqui ed using he (A) Zeb a ish and (B) Gu s Se ups. PE MPs subme ged in he aga ose gel appea ed as da k egions, isible as black spo s in
he da a. A
′
and B
′
p o ided a close iew. Selec ed MPs a e highligh ed wi h yellow a ows o indica e hei loca ion in he da ase .
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3.2. Ve i ica ion o pa icle size
To assess he accu acy o he mic oCT de ec ion o PE MPs, a com-
pa ison o he esul s om he e e ence samples wi h a pa icle size
analyse was pe o med. The de ec ed pa icles in he mic oCT da a
we e quan i ied, and hei size dis ibu ion was plo ed and compa ed
wi h he e e ence size dis ibu ion o he PE MP ba ch ob ained om a
pa icle size analyse be o e ga aging. This compa ison alida ed he
de ec ion accu acy o bo h mic oCT se ups, as desc ibed below.
The en i e size ange was success ully de ec ed using bo h CT se ups,
as illus a ed in Fig. 6A, B. The minimum size de ec ed emained
consis en ac oss bo h se ups and he pa icle size analyse . Howe e ,
pa icles la ge han 110 µm we e obse ed only in he Zeb a ish se up,
which was likely caused by clus e ing. The pa icles we e p one o
clus e o ma ion when hey we e wi hin 31 µm om each o he (Fig. 5),
which led o challenges in dis inguishing hei bounda ies h ough he
au oma ed segmen a ion due o minimal o no de ec able educ ion in
he in ensi y be ween adjacen pa icles. In his case, he Gu s Se up
helped o sepa a e he clus e ed pa icles, esul ing in he dec ease o
abundance o e 110
μ
m.
S a is ical es s we e conduc ed o e alua e di e ences be ween he
size dis ibu ion unc ions om mic oCT and he pa icle size analyse
Fig. 4. (A) De ec ed MPs in he da a acqui ed using he Zeb a ish se up. (B) Segmen ed pa icles a e highligh ed wi h blue ci cles, while pa icles no included in he
segmen a ion a e indica ed by ed a ows.
Fig. 5. Examples o segmen ed pa icles which ended o o m clus e s due o hei p oximi y (le ). Pa icles we e segmen ed manually (blue ci cles, igh ), allowing
o he ole ance o hei bounda ies. Dis ances be ween neighbou ing pa icles we e measu ed o de e mine how close we e he pa icles when hey s a ed
o clus e .
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(Fig. 6C, D). The wo-sample - es compa ed means, and he F- es
examined a iances o Gaussian dis ibu ions i ed o he da a om
each se up and he pa icle size analyse . The Zeb a ish Se up da a
(mean: 64, SD: 16) showed no signi ican di e ence in means (p =0.2)
bu a signi ican di e ence in a iances (p =0.01) compa ed o he
pa icle size analyse (mean: 61, SD: 11). Thus, while he a e age alues
we e simila , he sp ead o he Zeb a ish Se up da a was signi ican ly
di e en om ha o he dis ibu ion measu ed by he pa icle size
analyse .
In con as , he Gu s Se up da a (mean: 62, SD: 10) exhibi ed no
signi ican di e ences in ei he means (p =0.5) o a iances (p =0.3),
highligh ing hei close esemblance o he esul s o he pa icle size
analyse .
3.3. De ec ion o mic oplas ics in model issues
Bo h se ups om he p e iously desc ibed expe imen s on e e ence
samples, wi h he same sample p epa a ion s eps, we e u ilized o he
acquisi ion o model samples spiked wi h PE MPs: zeb a ish and
dissec ed gu s (Fig. 7A, B).
Di e en ly om he p e ious expe imen , s aining was inco po a ed
o enable he isualisa ion o he so issue and o consequen ly enhance
he con as be ween he issue and MPs (Fig. 7C, C’, D, D’).
To assess he e ec o s aining on he acqui ed da a, CNR and he
Fig. 6. Pa icle size dis ibu ion analysis compa ing mic oCT and pa icle size analyse measu emen – Zeb a ish and Gu s. CT measu emen s we e conduc ed on
e e ence samples using wo di e en CT se ups. (A) No malized size dis ibu ion o he Zeb a ish Se up, wi h he mic oCT da a ep esen ed as a his og am and he
pa icle size dis ibu ion o e laid as a cu e. (B) The co esponding size dis ibu ion o he Gu s Se up ollows he exac ep esen a ion. (C, D) Fi ed Gaussian
dis ibu ions compa ing mic oCT (blue) and pa icle size analyse ( ed) da a o he Zeb a ish and Gu s Se ups, espec i ely. (E) Resul s o he pa icle size analyse
wi h pa icle diame e s’ equency (ba s) and cumula i e numbe dis ibu ion (black line).
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dis ances be ween in ensi y his og am peaks de i ed om MP egions
and su ounding issue (aga ose gel in he case o e e ence samples)
we e calcula ed and summa ized in Table 2. O e all, CNR inc eased
a e s aining, and peak dis ances we e sligh ly b oade o s ained
zeb a ish and gu samples, indica ing enhanced con as be ween he
pa icles and he su ounding a ea.
Consequen ly, he segmen a ion o MPs was success ully pe o med
using he AI module o bo h ypes o he da a acqui ed wi h di e en CT
se ups. To a oid in oducing noise in o he segmen a ion, ca e was aken
o il e ou pa icles smalle han hose in he ac ual size dis ibu ion
ob ained om he pa icle size analyse (30 µm). A e wa ds, he il e ed
esul s we e analysed o e ie e he diame e s o he de ec ed pa icles.
In each sample, he gu s and he pa icles we e isualized using 3D
ende ing (Fig. 8A, B). Addi ionally, he pa icles we e colou -coded
based on hei diame e , and he clipping plane was applied om he
on al iew 3D ende o isualise he inside egions (Fig. 8A’, B’).
3.4. Mo phological and size analysis o mic oplas ics
Unde s anding whe e MPs accumula e could se e as a c ucial
pa ame e o co ela ing hei loca ions wi h speci ic diseases. The e-
o e, he numbe o pa icles de ec ed nea he illi egion whe e hey
could accumula e o e ime and po en ially ha m he o ganism, was
analysed. Addi ionally, he dis ance he pa icle a elled h ough he
gu was calcula ed o e eal i s mig a ion and he MP abundance was
calcula ed in h ee in es inal egions di ided in o an e io , middle and
pos e io .
The a io o he pa icles localized nea he illi compa ed o hose
close o he cen e o he gu e ealed ha only 25 % o he pa icles
we e loca ed in he cen al a ea, while he majo i y accumula ed nea
he illi, whe e hey we e mo e likely o a ach and agg ega e. The
majo i y o MPs we e ound in he an e io pa , ollowed by he middle
and pos e io egions o gu s, wi h 78 %, 18 %, and 4 % obse ed,
espec i ely.
Addi ionally, he passage o MPs h ough he gu was acked by
measu ing he dis ance om he beginning o he gu o he pa icle’s
loca ion. Fo ins ance, he posi ions o wo andomly selec ed pa icles
we e calcula ed o de e mine hei loca ions wi hin he colon. The i s
pa icle was ound a he end o he ascending colon, a 36 % o he o al
gu leng h, while he second pa icle was loca ed a 8 % o he o al
leng h, nea he beginning o he ascending colon (Fig. 9A).
I is impo an o no e ha he expe imen s we e conduc ed on model
zeb a ish ha we e manually spiked wi h MPs. The e o e, he esul s in
his sec ion p ima ily highligh he po en ial o mic oCT o MP
Fig. 7. Visualisa ion o (A) zeb a ish and (B) gu s scanned wi h mic oCT using wo selec ed se ups – Zeb a ish and Gu s. The c oss-sec ions display he loca ion o
inse ed pa icles wi hin he zeb a ish samples (C – ans e se plane, C’ – sagi al plane) and dissec ed gu s (D – ans e se plane, D’ – sagi al plane). Blue a ows
indica e he loca ion o MPs ( isible as black spo s), while yellow b acke s highligh he posi ion o he gu s.
Table 2
Quali y measu emen s o he selec ed sample da a and expe imen al se ups.
Zeb a ish Se up Gu s Se up
CNR
[dB]
In e -peak
dis ance
[g ay alues]
CNR
[dB]
In e -peak
dis ance
[g ay alues]
Aga 1.5 2273 0.1 3015
Model
sample
2.6 3488 1.34 3041
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de ec ion and he possibili ies o he da a analysis a he han ep e-
sen ing na u al biological condi ions.
4. Discussion
Despi e i s many ad an ages, mic oCT has no ye been widely used
in MP esea ch, which ypically elies on con en ional spec oscopic and
py oly ic me hods. Unlike adi ional app oaches, mic oCT allows o a
non-des uc i e analysis and o e s he capabili y o isualize in e nal
s uc u es in h ee dimensions. To da e, success ul applica ions o
mic oCT in MP de ec ion ha e been limi ed o geological s udies [30].
This s udy in oduced mic oCT as a no el me hod o de ec ing MPs
in biological samples. Using issue s aining echniques, in e nal s uc-
u es and MPs we e isualized, enabling he in es iga ion o MP accu-
mula ion wi hin o gans wi hou in asi e p ocedu es.
To assess he easibili y o mic oCT in his esea ch a ea, ini ial
expe imen s we e conduc ed on e e ence samples, whe e biological
issue was eplaced wi h aga ose gel o simpli y he design and MPs we e
success ully isualized. In he u he expe imen , MPs we e isualized
in he whole ish (Fish se up) and in he in es ine (Gu s se up). The en i e
size ange o MPs was success ully de ec ed wi h bo h op imized CT
se ups, and de ec ion accu acy was alida ed by compa ing hese esul s
o pa icle size analyse , showing dec eased accu acy using Zeb a ish
se up. The Gu s se up, in pa icula , demons a ed no s a is ical di e -
ence om he esul s ob ained by con en ional pa icle size analyse .
Howe e , achie ing he oxel size used in he Gu s se up equi es a
smalle sample, necessi a ing a ade-o be ween sample size and
desi ed de ec ion de ail when applying mic oCT o MP de ec ion in
biological samples. Following he success ul alida ion o mic oCT, ex-
pe imen s we e ex ended o model zeb a ish samples. He e, he s aining
applied o bo h he zeb a ish and gu samples o enhance so issue
isualiza ion imp o ed he MP de ec ion quali y.
Fig. 8. 3D ende ings o he segmen ed PE MPs in (A) zeb a ish and (B) gu samples. The gu samples we e segmen ed o an imp o ed isualiza ion. (A’, B’) F on al
iews and (A’’, B’’) ans e se iews we e cap u ed, wi h a clipping plane added o e eal he in e io o he samples. The clipping plane was posi ioned along he
midline o he sample and adjus ed acco ding o he selec ed iew.
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