Ci a ion: Rod íguez-Luna, M.R.;
Okamo o, N.; Al-Tahe , M.; Kelle ,
D.S.; Cinelli, L.; Hoske e Ashoka, A.;
Klymchenko, A.S.; Ma escaux, J.;
Diana, M. In Vi o Imaging
E alua ion o Fluo escence In ensi y
a Tail Emission o Nea -In a ed-I
(NIR-I) Fluo opho es in a Po cine
Model. Li e 2022,12, 1123. h ps://
doi.o g/10.3390/li e12081123
Academic Edi o s: Joos R. an de
Vo s and Alexande Vah meije
Recei ed: 12 July 2022
Accep ed: 25 July 2022
Published: 27 July 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
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li e
A icle
In Vi o Imaging E alua ion o Fluo escence In ensi y a Tail
Emission o Nea -In a ed-I (NIR-I) Fluo opho es in
a Po cine Model
Ma ía Ri a Rod íguez-Luna 1,2,* , Na iaki Okamo o 1,2 , Mahdi Al-Tahe 1,3 , Debo ah S. Kelle 4,
Lo enzo Cinelli 5, Anila Hoske e Ashoka 6, And ey S. Klymchenko 6, Jacques Ma escaux 1
and Michele Diana 1,2
1Resea ch Ins i u e agains Diges i e Cance (IRCAD), 1 Place de l’Hôpi al, 67000 S asbou g, F ance;
[email p o ec ed] (N.O.); mahdi.al- ahe @i cad. (M.A.-T.); [email p o ec ed] (J.M.);
[email p o ec ed] (M.D.)
2ICube Labo a o y, Pho onics Ins umen a ion o Heal h, 67081 S asbou g, F ance
3Maas ich Uni e si y Medical Cen e , 6229 Maas ich , The Ne he lands
4Ma ks Colo ec al Su gical Associa es, Lankenau Medical Cen e , Main Line Heal h,
Wynnewood, PA 19096, USA; [email p o ec ed]
5Depa men o Gas oin es inal Su ge y, San Ra aele Hospi al IRCCS, 20132 Milan, I aly;
lo enzo.cinelli@i cad.
6Labo a oi e de Bioimage ie e Pa hologies, UMR 7021 CNRS, Uni e si éde S asbou g, 74 Rou e du Rhin,
67401 Illki ch, F ance; anil.hoske [email p o ec ed] (A.H.A.); and ey[email p o ec ed] (A.S.K.)
*Co espondence: i a. [email p o ec ed]
Abs ac :
O e he las decade luo escence-guided su ge y has been p ima ily ocused on he NIR-I
window. Howe e , he NIR-I window has cons ain s, such as limi ed pene a ion and sca e ing.
Consequen ly, explo ing he pe o mance o NIR-I dyes a longe wa eleng hs (i.e., he NIR-II window)
is c ucial o expanding i s applica ion. Two luo opho es we e used in h ee pigs o iden i y he
mean luo escence in ensi y (MFI) using wo comme cially a ailable NIR-I and NIR-II came as. The
nea -in a ed coa ing o equipmen (NICE) was used o iden i y endoluminal su gical ca he e s and
indocyanine g een (ICG) o common bile duc (CBD) cha ac e iza ion. The NIR-II window e alua ion
showed an MFI o 0.4 a bi a y uni s (a.u.)
±
0.106 a.u. in small bowel NICE-coa ed ca he e s and an MFI
o 0.09 a.u.
±
0.039 a.u. in gas ic ones. In CBD cha ac e iza ion, he ICG MFI was 0.12 a.u.
±
0.027 a.u.,
0.18 a.u.
±
0.100 a.u., and 0.22 a.u.
±
0.041 a.u. a 5, 35, and 65 min, espec i ely. This
in i o
imaging
e alua ion o NIR-I dyes con i ms i s applica ion in he NIR-II domain. To he bes o ou knowledge,
his is he i s s udy assessing he MIF o NICE in he NIR-II window using a comme cially a ailable
sys em. Fu he compa a i e ials a e necessa y o de e mine he supe io i y o NIR-II imaging sys ems.
Keywo ds:
nea -in a ed window I (NIR-I); nea -in a ed window II (NIR-II); luo escence imaging
sys em; bile duc imaging; coa ed medical ma e ials; indocyanine g een; ICG; luo escence image-
guided su ge y; FIGS
1. In oduc ion
Fluo escence image-guided su ge y (FIGS) is a con inuously e ol ing ield. An in-
c easing numbe o ad anced echnological imaging sys ems a e being in oduced o open,
lapa oscopic, and obo ic su ge y wi h he goal o inc easing ope a i e p ecision [
1
–
3
].
One ool used in his a ea is nea -in a ed (NIR) luo escence imaging (FI) echnology.
NIR-FI allows o a eal- ime ideo a e isualiza ion wi h a highe con as and deepe
pene a ion as compa ed o he isible ligh spec um, allowing su geons o be e disce n
ana omical s uc u es. NIR-FI equi es he adminis a ion o a luo escen con as agen [
4
].
Essen ially,
in i o
NIR bioimaging can be unde s ood in he con ex o ligh p opaga ion.
As desc ibed by F angioni e al., a pho on a els h ough issue o each he luo escen
Li e 2022,12, 1123. h ps://doi.o g/10.3390/li e12081123 h ps://www.mdpi.com/jou nal/li e
Li e 2022,12, 1123 2 o 9
con as agen , and a ious esul s may occu depending on issue sca e , aniso opy, and
e lec ance index. The same happens o he pho on emi ed by luo opho e. To exci e and
de ec he spec al signal, he use o an op ical imaging sys em is equi ed [5] (Figu e 1).
Li e 2022, 12, x FOR PEER REVIEW 2 o 10
p opaga ion. As desc ibed by F angioni e al., a pho on a els h ough issue o each he
luo escen con as agen , and a ious esul s may occu depending on issue sca e , an-
iso opy, and e lec ance index. The same happens o he pho on emi ed by luo opho e.
To exci e and de ec he spec al signal, he use o an op ical imaging sys em is equi ed
[5] (Figu e 1).
Figu e 1. Schema ic o e iew o he use o NIRF in su ge y, classical iew when using NIR-I imag-
ing sys em.
O e he pas decade, FIGS has been mos ly dedica ed o wha is known as he NIR-
I window, which co esponds o a wa eleng h o 700 o 900 nm in he elec omagne ic
spec um [4,6]. Howe e , i s pe o mance in li ing issues has some limi a ions, such as
ligh abso p ion and loss o pho ons due o sca e ing. Indeed, hese d awbacks inc ease
exponen ially wi h dep h, allowing o issue pene a ion o less han 1 cm, which es ic s
he NIR-I window capaci y om de ec ing deepe ana omical ea u es [7].
The i s de eloped luo opho e, which gained Food and D ug Adminis a ion
(FDA) app o al o clinical use, was indocyanine g een (ICG) [8]. ICG is a NIR-I dye,
which co esponds o a peak luo escen emission wa eleng h in he λ
ex
max = 800 nm,
λ
em
max = 820 nm window. Since i binds igh ly o plasma p o eins, i s hal -li e is 18 min,
which is one o he main easons o i s ex ensi e adop ion as a na iga ional ool o assess
supe icial pe usion [9]. Due o i s mos ly hepa ic clea ance, ICG is also used o e alua e
li e unc ion and o cha ac e ize bilia y ana omy. Lympha ic d ainage mapping du ing
oncological esec ions and umo al ma gin ecogni ion has also been desc ibed in he ep-
e oi e o applica ions [7].
ICG has no only been he mos clinically used luo opho e [10] bu i has also se ed
as a co ne s one in he enginee ing o NIR imaging sys ems, especially hose e alua ing
he NIR-I window. Recen ly, he ICG luo escen emission spec a in he NIR-II window
also e e ed as he “ ail emission” o “o -peak NIR-I luo escence emission” ha e been
desc ibed [11]. In an in i o small animal s udy, S a osolski e al. con i med ICG’s ap-
plicabili y in quan i a i e bioimaging assessmen in he NIR II-window using a cus om-
made InGaAS came a [12].
Figu e 1.
Schema ic o e iew o he use o NIRF in su ge y, classical iew when using NIR-I
imaging sys em.
O e he pas decade, FIGS has been mos ly dedica ed o wha is known as he NIR-I
window, which co esponds o a wa eleng h o 700 o 900 nm in he elec omagne ic
spec um [
4
,
6
]. Howe e , i s pe o mance in li ing issues has some limi a ions, such as
ligh abso p ion and loss o pho ons due o sca e ing. Indeed, hese d awbacks inc ease
exponen ially wi h dep h, allowing o issue pene a ion o less han 1 cm, which es ic s
he NIR-I window capaci y om de ec ing deepe ana omical ea u es [7].
The i s de eloped luo opho e, which gained Food and D ug Adminis a ion (FDA)
app o al o clinical use, was indocyanine g een (ICG) [
8
]. ICG is a NIR-I dye, which co e-
sponds o a peak luo escen emission wa eleng h in he
λex
max = 800 nm,
λem max = 820 nm
window. Since i binds igh ly o plasma p o eins, i s hal -li e is 18 min, which is one o he
main easons o i s ex ensi e adop ion as a na iga ional ool o assess supe icial pe usion [
9
].
Due o i s mos ly hepa ic clea ance, ICG is also used o e alua e li e unc ion and o cha -
ac e ize bilia y ana omy. Lympha ic d ainage mapping du ing oncological esec ions and
umo al ma gin ecogni ion has also been desc ibed in he epe oi e o applica ions [7].
ICG has no only been he mos clinically used luo opho e [
10
] bu i has also se ed
as a co ne s one in he enginee ing o NIR imaging sys ems, especially hose e alua ing
he NIR-I window. Recen ly, he ICG luo escen emission spec a in he NIR-II window
also e e ed as he “ ail emission” o “o -peak NIR-I luo escence emission” ha e been
desc ibed [
11
]. In an
in i o
small animal s udy, S a osolski e al. con i med ICG’s applica-
bili y in quan i a i e bioimaging assessmen in he NIR II-window using a cus om-made
InGaAS came a [12].
The goal o his p eclinical la ge animal s udy is o e alua e he “ ail emission” o wo
NIR-I luo opho es, namely ICG and nea -in a ed coa ing o equipmen (NICE), using a
quan i a i e assessmen wi h mean luo escence in ensi y (MFI) in he NIR-II window. Fo
his pu pose, ICG was in a enously (IV) adminis e ed as a con as agen o cha ac e ize
he common bile duc (CBD) and NICE was used as a ca he e coa ing agen .
Li e 2022,12, 1123 3 o 9
2. Ma e ials and Me hods
The s udy was conduc ed in s ic acco dance wi h he ecommenda ions published in
he Guide o he Ca e and Use o Labo a o y Animals o he Na ional Ins i u es o Heal h.
The p esen s udy is pa o he ELIOS p o ocol (Endoscopic Luminescen Imaging o On-
cology Su ge y), ully app o ed by he local E hical Commi ee on Animal Expe imen a ion
(ICOMETH No. 38.2016.01.085), and by he F ench Minis y o Supe io Educa ion and
Resea ch (MESR) (APAFIS#8721-2017013010316298- 2). All sec ions o his epo adhe e
o he ARRIVE Guidelines o epo ing animal esea ch [13].
This expe imen al s udy included h ee pigs (Sus sc o a domes icus, ssp. La ge Whi e,
bo h gende s, mean weigh : 32
±
4.54 kg). The 3Rs p inciple ( eplacemen , e inemen , and
educ ion) we e ollowed in compliance wi h he op imal animal wel a e condi ions [14].
Animals as ed o 24 h be o e su ge y. They ecei ed an in amuscula (IM) injec ion o
Zolazepam + Tile amine 10 mg/kg (Zole il ND, Vi bac, F ance) as p emedica ion. Anes hesia
was induced by means o an IV injec ion o P opo ol 3 mg/kg (P opo ol Lipu o ND, B B aun,
F ance) + Rocu onium 0.8 mg/kg (Esme on ND, MSD, F ance), hence allowing o in uba ion
and mechanical en ila ion. Pigs we e seda ed du ing he expe imen ia an inhala ion o
iso lu ane 2–3% (Iso lu in ND, Axience F ance) + Oxygen. Bup eno phine (Bup eca e ND,
Axience, F ance) a 0.01 mg/kg, adminis e ed in amuscula ly, was used as a painkille . A
he end o he expe imen s, he pigs we e eu hanized unde deep anes hesia (iso lu ane 5%)
wi h a le hal IV injec ion o Pen oba bi al 40 mg/kg (Exagon ND, Axience, F ance).
2.1. ICG Dye P epa a ion
ICG (In acyanine
®
, Se b labo a o ies, Pa is, F ance) was p epa ed acco ding o he
manu ac u e ’s speci ica ions, dilu ing he powde in 5% glucose solu ion o a inal dilu ion
o 25 mg/10 mL. The ICG dose was calcula ed acco ding o he animal’s weigh (0.10 mg/kg).
2.2. Fluo escen NICE-Coa ed Ca he e P epa a ion
The nea -in a ed coa ing o equipmen (NICE) was syn hesized by combining a bio-
compa ible polyme poly (me hyl me hac yla e) (PMMA) wi h a speci ically enginee ed
luo escen dye [
15
]. NICE is an ul ab igh and s able biocompa ible luo escen coa ing
ha can coa su gical de ices o any ma e ial and be isualized using comme cially a ail-
able su gical NIR came as. NICE displays a spec al ange simila o ICG and a 15–20- old
highe luo escence signal. Addi ionally, he pho os abili y a e 1 h i adia ion a 760 nm
only has a mino dec ease in luo escence in ensi y. To da e, NICE has been alida ed in
mul iple p eclinical models [15–19].
Two echniques o coa medical de ices (i.e., dip coa ing and pain b ushing) ha e been
published by ou g oup [
15
]. In his s udy, we use he dip-coa ing me hod o coa 16 F ench
ca he e s, which consis s o h ee imme sions o he ca he e in o he NICE wi h a pe iod o
d ying be ween each imme sion (coa ing ime: oughly 1 h).
Fluo escence in ensi y o he coa ings we e measu ed a oom empe a u e on an Edin-
bu g FS5 spec o luo ome e . A polyme coa ing wi hou dye was used as a baseline o all
he measu emen s. NIR luo escence images o he coa ings we e cap u ed wi h he help o a
home-made NIR image se up equipped wi h a scien i ic g ade sCMOS came a, 740 nm LED
ligh sou ce, and 835/70 nm band pass il e . Fluo escence images we e analyzed and he
luo escence in ensi y was quan i ied wi h he help o ImageJ so wa e (Figu e 2).
2.3. Ope a ing Se up and Expe imen al Wo k low
Animal Model
A median lapa o omy was pe o med. A po ion o he small bowel (namely he
jejunum) was hen selec ed 200 cm away om he ligamen o T ei z, ollowed by an
en e o omy. A e he in oduc ion o he coa ed ca he e , he en y hole was hen closed
wi h 3/0 polyso b in a unning su u e ashion. The ligh s o he ope a ing oom we e u ned
o du ing imaging acquisi ion in o de o educe ligh in e e ence. The image cap u e
was pe o med h ough pho os, i s using he NIR-I imaging sys em, hen ollowed by he
Li e 2022,12, 1123 4 o 9
NIR-II imaging sys em (acquisi ion ime: oughly 60 s o each de ice). As o he NIR-I
came a, a ze o-deg ee lapa oscope D-ligh TH102 came a H3-Z FI TC300 CCU (KARL
STORZ, GmbH, Ge many) was used as a con ol o he NIR-II came a KIS II (Kae Labs,
Nan es, F ance). The came a’s dis al lens was placed a an app oxima ely 20 cm dis ance
o cap u e he en i e su gical a ea o in e es . A calib a ion aid, p o iding a cons an
luo escence signal (G een Balance
™
ICG Re e ence Ca d; Diagnos ic G een; Aschheim–
Do nach, Ge many), was placed in he abdominal ca i y, close he egion o in e es o
p o ide a e e ence luo escence alue. This e e ence ca d was he e o e used o compu e
he luo escence in ensi y as a bi a y uni s (a.u.) o co ec any po en ial dis ance bias.
Li e 2022, 12, x FOR PEER REVIEW 4 o 10
analyzed and he luo escence in ensi y was quan i ied wi h he help o ImageJ so wa e
(Figu e 2).
Figu e 2. Nea -in a ed coa ing o equipmen . (a) Emission esponse o he coa ings wi h a ying
concen a ions o dye. (b) Analysis o luo escence in ensi y a e each coa ing.
2.3. Ope a ing Se up and Expe imen al Wo k low
Animal Model
A median lapa o omy was pe o med. A po ion o he small bowel (namely he je-
junum) was hen selec ed 200 cm away om he ligamen o T ei z, ollowed by an en e -
o omy. A e he in oduc ion o he coa ed ca he e , he en y hole was hen closed wi h
3/0 polyso b in a unning su u e ashion. The ligh s o he ope a ing oom we e u ned o
du ing imaging acquisi ion in o de o educe ligh in e e ence. The image cap u e was
pe o med h ough pho os, i s using he NIR-I imaging sys em, hen ollowed by he
NIR-II imaging sys em (acquisi ion ime: oughly 60 s o each de ice). As o he NIR-I
came a, a ze o-deg ee lapa oscope D-ligh TH102 came a H3-Z FI TC300 CCU (KARL
STORZ, GmbH, Ge many) was used as a con ol o he NIR-II came a KIS II (Kae Labs,
Nan es, F ance). The came a’s dis al lens was placed a an app oxima ely 20 cm dis ance
o cap u e he en i e su gical a ea o in e es . A calib a ion aid, p o iding a cons an luo-
escence signal (G een Balance™ ICG Re e ence Ca d; Diagnos ic G een; Aschheim–Do -
nach, Ge many), was placed in he abdominal ca i y, close he egion o in e es o p o-
ide a e e ence luo escence alue. This e e ence ca d was he e o e used o compu e
he luo escence in ensi y as a bi a y uni s (a.u.) o co ec any po en ial dis ance bias.
The NIR-I came a has a s anda d whi e ligh obse a ion ha can be swi ched o an
NIR-I mode using a oo swi ch pedal, which concu en ly ac i a es he NIR-I ligh sou ce
and a bypass il e . The NIR-II came a used was he NIR-II KIS II op ical imaging sys em,
which is designed o an open su gical se ing. I wo ks wi h a compu e -based in e ace,
which allows o he con ol o echnical pa ame e s, such as exposu e ime. Addi ionally,
il e s can be moun ed on o he dis al end o he lens, es ic ing he wa eleng h o he
shu e . Fo his expe imen , a 1200 nm il e was used wi h a p ese 60 ms exposu e ime.
Simila o he image acquisi ion o he small bowel, a cold gas o omy was pe o med
a he pos e io gas ic wall. The gas ic luid was aspi a ed p io o ca he e inse ion,
p e en ing image in e e ence and ca he e mig a ion. As in he small bowel, he gas o-
omy was closed using 3/0 polyso b. The pa e n o image acquisi ion o NIR-I and NIR-
II came as was p e iously desc ibed.
Fo he bilia y p ocedu e, he assis an su geon exposed he CBD by e ac ing he
gallbladde la e ally. The su geon hen dissec ed he dis al pa o he CBD o ensu e i s
p ope skele oniza ion. A e an adequa e CBD dissec ion, he anes hesiologis in a e-
nously in used he p epa ed ICG solu ion in o a pe iphe al enous line. A 20 mL bolus o
saline solu ion 0.9% was adminis e ed o ensu e he ICG eached he sys emic enous
blood (Figu e 3).
Figu e 2.
Nea -in a ed coa ing o equipmen . (
a
) Emission esponse o he coa ings wi h a ying
concen a ions o dye. (b) Analysis o luo escence in ensi y a e each coa ing.
The NIR-I came a has a s anda d whi e ligh obse a ion ha can be swi ched o an
NIR-I mode using a oo swi ch pedal, which concu en ly ac i a es he NIR-I ligh sou ce
and a bypass il e . The NIR-II came a used was he NIR-II KIS II op ical imaging sys em,
which is designed o an open su gical se ing. I wo ks wi h a compu e -based in e ace,
which allows o he con ol o echnical pa ame e s, such as exposu e ime. Addi ionally,
il e s can be moun ed on o he dis al end o he lens, es ic ing he wa eleng h o he
shu e . Fo his expe imen , a 1200 nm il e was used wi h a p ese 60 ms exposu e ime.
Simila o he image acquisi ion o he small bowel, a cold gas o omy was pe o med
a he pos e io gas ic wall. The gas ic luid was aspi a ed p io o ca he e inse ion,
p e en ing image in e e ence and ca he e mig a ion. As in he small bowel, he gas o omy
was closed using 3/0 polyso b. The pa e n o image acquisi ion o NIR-I and NIR-II
came as was p e iously desc ibed.
Fo he bilia y p ocedu e, he assis an su geon exposed he CBD by e ac ing he
gallbladde la e ally. The su geon hen dissec ed he dis al pa o he CBD o ensu e
i s p ope skele oniza ion. A e an adequa e CBD dissec ion, he anes hesiologis in a-
enously in used he p epa ed ICG solu ion in o a pe iphe al enous line. A 20 mL bolus
o saline solu ion 0.9% was adminis e ed o ensu e he ICG eached he sys emic enous
blood (Figu e 3).
The i s NIR image acquisi ion was achie ed a e 5 min ( 5) o injec ion. The wo NIR
imaging sys ems we e used as p e iously desc ibed. Fu he acquisi ions we e pe o med
e e y 30 min o 1 h ( 35 and 65). As o he endoluminal NICE-coa ed ca he e s, he
ex e nal ligh in e e ence was a oided du ing image acquisi ion. A e 65, a he end o
he p ocedu e, biopsies we e aken o his opa hological analysis.
Du ing all expe imen s, he ICG e e ence ca d was in he ope a i e ield nex o he
egion o in e es o compu e he no malized luo escence a e wa ds.
Li e 2022,12, 1123 5 o 9
Li e 2022, 12, x FOR PEER REVIEW 5 o 10
Figu e 3. Visualiza ion o he endoluminal small bowel NICE-coa ed ca he e and CBD cha ac e i-
za ion a 35 min a e ICG adminis a ion; (A) small bowel RGB image; (B) small bowel NIR-I win-
dow, o e lay image (RGB + luo escence); (C) small bowel NIR-II window, o e lay (RGB + luo es-
cence) (D) CBD RGB image; (E) CBD NIR-I window, o e lay image (RGB + luo escence); (F) CBD
NIR-II window, o e lay (RGB + luo escence). Abb e ia ions: RGB— ed, g een, blue; NIR—nea -
in a ed; NICE—nea -in a ed coa ing o equipmen , CBD—common bile duc , ICG—indocyanine
g een.
The i s NIR image acquisi ion was achie ed a e 5 min ( 5) o injec ion. The wo
NIR imaging sys ems we e used as p e iously desc ibed. Fu he acquisi ions we e pe -
o med e e y 30 min o 1 h ( 35 and 65). As o he endoluminal NICE-coa ed ca he e s,
he ex e nal ligh in e e ence was a oided du ing image acquisi ion. A e 65, a he end
o he p ocedu e, biopsies we e aken o his opa hological analysis.
Du ing all expe imen s, he ICG e e ence ca d was in he ope a i e ield nex o he
egion o in e es o compu e he no malized luo escence a e wa ds.
2.4. Pos -P ocessing Fluo escence Analyses
Mean luo escen in ensi ies we e calcula ed a e he expe imen al analysis as o al
coun s pe ROI pixel. The OSIRIX Li e 12.0.1 imaging so wa e (Pixmeo, Gene a, Swi -
ze land) was used o quan i y luo escence in ensi y. We co ec ed he luo escence by
compu ing he ela i e luo escence in ensi y as he a io be ween he a ge and he adja-
cen e e ence ca d’s luo escence in ensi y. As p e iously desc ibed, luo escence in en-
si y was de ined in a.u. A eas wi h ligh sca e ing we e a oided as ROI.
2.5. S a is ical Analysis
Da a we e collec ed and eco ded in a comme cially a ailable da abase (Excel
sp eadshee in e sion 2021, Mic oso Co po a ion, Redmond, WA, USA) o subsequen
s a is ical analysis. Da a we e epo ed as means and s anda d de ia ions (SD) unless o h-
e wise indica ed. S a is ical compa isons be ween a iables we e made using a S uden ’s
- es o con inuous o disc e e a iables. A p- alue < 0.05 was conside ed s a is ically
signi ican .
Figu e 3.
Visualiza ion o he endoluminal small bowel NICE-coa ed ca he e and CBD cha ac e iza-
ion a 35 min a e ICG adminis a ion; (
A
) small bowel RGB image; (
B
) small bowel NIR-I window,
o e lay image (RGB + luo escence); (
C
) small bowel NIR-II window, o e lay (RGB + luo escence)
(
D
) CBD RGB image; (
E
) CBD NIR-I window, o e lay image (RGB + luo escence); (
F
) CBD NIR-II
window, o e lay (RGB + luo escence). Abb e ia ions: RGB— ed, g een, blue; NIR—nea -in a ed;
NICE—nea -in a ed coa ing o equipmen , CBD—common bile duc , ICG—indocyanine g een.
2.4. Pos -P ocessing Fluo escence Analyses
Mean luo escen in ensi ies we e calcula ed a e he expe imen al analysis as o-
al coun s pe ROI pixel. The OSIRIX Li e 12.0.1 imaging so wa e (Pixmeo, Gene a,
Swi ze land) was used o quan i y luo escence in ensi y. We co ec ed he luo escence
by compu ing he ela i e luo escence in ensi y as he a io be ween he a ge and he
adjacen e e ence ca d’s luo escence in ensi y. As p e iously desc ibed, luo escence
in ensi y was de ined in a.u. A eas wi h ligh sca e ing we e a oided as ROI.
2.5. S a is ical Analysis
Da a we e collec ed and eco ded in a comme cially a ailable da abase (Excel sp ead-
shee in e sion 2021, Mic oso Co po a ion, Redmond, WA, USA) o subsequen s a is ical
analysis. Da a we e epo ed as means and s anda d de ia ions (SD) unless o he wise
indica ed. S a is ical compa isons be ween a iables we e made using a S uden ’s - es o
con inuous o disc e e a iables. A p- alue < 0.05 was conside ed s a is ically signi ican .
3. Resul s
The p ima y esul s a e summa ized in Table 1. In all animals, we we e able o de ec
he MFI o endoluminal NICE-coa ed ca he e s and ICG du ing CBD cha ac e iza ion a
di e en ime poin s using he NIR-II imaging sys em.
Li e 2022,12, 1123 6 o 9
Table 1.
Fluo escen signal in ensi y exp essed as a bi a y uni s (a.u.) iden i ying endoluminal
NICE-coa ed ca he e s and CBD cha ac e iza ion a e ICG adminis a ion.
NIR-I Window NIR-II Window pValue
Endoluminal
NICE-coa ed
ca he e s in he
small bowel
0.34 ±0.007 a.u. 0.14 ±0.106 a.u. p= 0.111
Endoluminal
NICE-coa ed
ca he e s in he
s omach
0.26 ±0.156 a.u. 0.09 ±0.039 a.u. p= 0.248
5
0.52 ±0.198 a.u. 0.12 ±0.027 a.u. p= 0.101
CBD a e
in a enous ICG
injec ion
35
0.54 ±0.216 a.u 0.18 ±0.100 a.u p= 0.129
65
0.62 ±0.251 a.u. 0.22 ±0.041 a.u. p= 0.151
Abb e ia ions: NIR—nea -in a ed, CBD—common bile duc , NICE—nea -in a ed coa ing o equipmen .
All su gical p ocedu es we e pe o med wi hou any ad e se e en s.
4. Discussion
The ongoing esea ch in op ical imaging has been mos ly dedica ed o NIR-I (~700–900 nm).
The p esen s udy analyzed he easibili y o NIR-I luo escen dye bioimaging in he NIR-II
window (1000–1700 nm) [
10
]. We we e able quan i y he luo escence in ensi y o ICG and NICE
a o -peak spec a, con i ming hei ail emission.
The NIR-II window has ecen ly ga ne ed a en ion since i is well-known ha , a a longe
wa eleng h a e lase exci a ion, pho ons a el in he issues wi h signi ican ly less a enua ion
and sca e ing. The sca e ing o NIR-II pho ons (scaling wi h
λ
-
α
;
α
= 0.2–4 o mos issues) is
he e o e diminished, he eby p o iding a highe and mo e p ecise esolu ion [
11
]. To op imize
FIGS wi hin he NIR-II window, de elopmen s o co ne s one componen s such as luo opho es
and op ical imaging sys ems mus be encou aged.
As o luo opho es, NIR-II nanoma e ials ha e ecen ly been de eloped (single-
walled ca bon nano ubes, quan um do s, and a e-ea h-doped nanopa icles). Fo ins ance,
Sa anko e al. demons a ed high quan um yield and an ioxidan ac i i y in a sus ainable
biomass was e model ha enabled ion sensing and cellula imaging o cance cells [20].
Howe e , he la es NIR-II o ganic dyes ha e mos ly implemen ed a cyanine o elec-
on dono -accep o -dono (CH1055PEG ~1000–1700 nm) s uc u e [
21
,
22
] in which he
ino ganic p ope ies a e o conce n o clinical sa e y, since hese ma e ials can be accu-
mula ed wi hin solid o gans, such as he li e o spleen [
10
]. Consequen ly, NIR-II dyes
a e s ill in a de elopmen phase, and he e a e cu en ly no NIR-II dyes which ha e been
FDA-app o ed o clinical use.
As a esul , he e is a signi ican in e es in e alua ing alida ed NIR-I dyes p o en o
achie e a high quan um yield (QY) pe o mance in he NIR-II window [11].
As p e iously desc ibed, ICG ail emission was unin en ionali y ound
in i o
[
17
]. I
was la e on s udied by S a osolski e al. in an ICG pe usion s udy in mice models [
12
]. In
he p eclinical s udy, au ho s used a cus om-buil spec al NIR assembly o simul aneously
assess imaging o he wo NIR windows. Ta ge - o-backg ound a ios (TBRs) in he NIR-II
imaging sys em almos double hose o he NIR-I imaging sys em [12].
Siqi Gao e al. ecen ly published a la ge animal s udy using a clinically a ailable and
imp o ed NIR-I/II mul ispec al imaging sys em. Au ho s simul aneously assessed ICG
a 800 nm and 1100 nm wa eleng hs o map he mic o ascula ne wo k and assess he
pe usion s a us on a skin a ulsion–inju y model [23].
Li e 2022,12, 1123 7 o 9
They used an upg aded comme cially a ailable imagining sys em ha was used
in he i s in-human ICG-guided li e esec ion o p ima y and me as a ic li e umo s
in 23 pa ien s in 2019. This s udy ound ha NIR-II imaging p o ided a highe umo -
de ec ion sensi i i y (100% s. 90.6%), a highe umo o no mal li e issue signal a io
(5.33 s. 1.45) and an enhanced umo de ec ion a e (56.41% s. 46.15%) [24].
These pi o al epo s ha e p o ided a plausible ounda ion o ou cu en expe imen ,
which de ec s he ICG MFI a he ail emission (1200 nm) in a la ge animal model using
a ecen comme cially a ailable NIR-II imaging sys em. In e es ingly, he p e iously
published NIR-II o -peak emission o ICG has e en been de ec able up o 1575 nm [17].
By assessing ICG luo escence in ensi y a he ail emission, we we e able o cha ac e -
ize he CBD a 5, 35, and 65 min. Failu e o iden i y he ana omy seems o be one o he main
ac o s ela ed o bile duc inju ies (BDIs), i.e., clinical scena ios o ch onic in lamma ion [
4
].
FIGS in he NIR-I window has been as ly s udied, he eby helping su geons p e en
BDIs [
25
]. Despi e he e o s made as well as he in oduc ion o ad anced echnology in
his domain, including a i icial in elligence, he incidence o BDIs emains s able. Fu u e
esea ch in hickened in lamed issue aiming o enhance FIGS in he NIR-II window is a
subjec o eal in e es [26].
Fluo opho es, which can adhe e o su gical ma e ials, a e a ac i e op ions as hey
do no necessi a e any sys emic adminis a ion, hence pe mi ing as e clinical ansla-
ion. NICE was de eloped as a coa ing dye al e na i e o ICG. Ou esea ch g oup has
p e iously ob ained excellen esul s in e ms o a cons an spec al signal while expe i-
men ing in p eclinical models. To da e, NICE-coa ed medical de ices ha e been used o
in aope a i ely iden i y he u e e and he u e h a [
19
] o gas oin es inal clip ma king
(o e - he-scope clips (OVESCO)) [
14
] and o guide magne ic anas omo ic de ices du ing
endolapa oscopic gas ojejunos omies [
18
]. Ou p e ious s udies e alua ing NICE ha e
gene ally been pe o med wi hin he NIR-I window. Since NICE is a cyanine 7.5 de i a e
and as he emission wa eleng h is compa able o ICG, i was app op ia e o e alua ing
NICE pe o mance in he NIR-II domain [16].
Compa able o CBD cha ac e iza ion, endoluminal NICE-coa ed ca he e s in he small
bowel and he s omach we e isualized in all animals using he NIR-II imaging sys em,
hence illus a ing NICE luo escence in ensi y a he ail emission. In eali y, NICE achie es
a supe io b igh ness (15–20- old highe ) compa ed o ICG [
16
]. The nex sensible s ep in
esea ch would be o e alua e NICE wi hin deepe ana omical issues.
The NIR-II window is s ill in i s in ancy compa ed o he as e olu ion ound in NIR-I
bioimaging. Con a y o NIR-I came as, cha ge-coupled de ices (CCD) canno be used in
NIR-II imaging sys ems since hey a e la gely insensi i e o wa eleng hs o e 1000 nm [
26
].
Consequen ly, NIR-II sys ems equi e he use o mo e sophis ica ed and highe -cos de ec o s,
such as a compound semiconduc o build-up o InGaAs o HgCdTe [
27
]. A p esen , he
majo i y o NIR-II imaging sys ems ha e been enginee ed o luo escence mic oscopy, some
o hem cus omized o p eclinical s udies, and e y ew a e a ailable in clinical se ings [
11
].
The me i o ou s udy lies in he ac ha his p eclinical ial e alua es he luo escence
in ensi y a he ail emission o wo NIR-I dyes. While cha ac e izing ana omical s uc u es
o in e es in all po cine models, ICG and NICE p o e o ha e adequa e pe o mance in he
NIR-II domain.
The limi a ion o ou s udy is ep esen ed by he small sample size. In addi ion,
al hough i was beyond he scope o he s udy’s objec i e, we did no ind any supe io i y
when compa ing he wo NIR imaging sys ems, which ha e been claimed by he p e ious
NIR-II ex i o and
in i o
s udies, as well as o he clinical ials. We acknowledge ha da a
ha e been collec ed om di e en ana omical si es, whe e di e ences in issue s uc u e
could a ec he yield o he emission signal in he wo NIR-I and NIR-II windows; he e o e
he esul s should be conside ed wi h cau ion.
Li e 2022,12, 1123 8 o 9
5. Conclusions
The
in i o
assessmen o ICG and NICE luo escence in ensi y a he ail emission is
easible, hence allowing o CBD disc imina ion and iden i ica ion o endoluminal NICE-
coa ed su gical ca he e s in a la ge animal model. This s udy suppo s he p e iously
epo ed accu a e
in i o
pe o mance o ICG in he NIR-II window a longe wa eleng hs
(1200 nm), and i ep esen s NICE assessmen in he NIR-II domain o he i s ime,
p o iding a ounda ion o u he p eclinical con i ma o y ials.
Au ho Con ibu ions:
M.R.R.-L., M.A.-T. and M.D. designed he s udy. M.R.R.-L., N.O. and L.C.
conduc ed he expe imen s. A.S.K. and M.D. a e he in en o s o he NICE coa ing. A.H.A. syn he-
sized he luo escen coa ing equi ed o he expe imen s. M.R.R.-L., N.O. and M.A.-T. analyzed he
da a. M.R.R.-L., M.A.-T. and D.S.K. d a ed he manusc ip . J.M., D.S.K., A.H.A. and A.S.K. e ised
he manusc ip o majo in ellec ual con en . All au ho s ha e ead and ag eed o he published
e sion o he manusc ip .
Funding:
This s udy was unded by he ARC Founda ion o Cance Resea ch, a F ench ounda ion
en i ely dedica ed o cance esea ch, in he amewo k o a la ge p ojec known as ELIOS (Endo-
scopic Luminescen Imaging o P ecision Oncologic Su ge y), and was aimed a he de elopmen
o luo escence-guided su ge y (h ps://www. onda ion-a c.o g/p oje s/amelio e -diagnos ic-e -
ai emen -chi u gical-cance s-diges i s (accessed on 24 July 2022)).
Ins i u ional Re iew Boa d S a emen :
The s udy was conduc ed in s ic acco dance wi h he ec-
ommenda ions published in he Guide o he Ca e and Use o Labo a o y Animals o he Na ional
Ins i u es o Heal h. The p esen s udy is pa o he ELIOS p o ocol (Endoscopic Luminescen Imag-
ing o Oncology Su ge y), ully app o ed by he local E hical Commi ee on Animal Expe imen a ion
(ICOMETH No. 38.2016.01.085), and by he F ench Minis y o Supe io Educa ion and Resea ch
(MESR) (APAFIS#8721-2017013010316298- 2).
In o med Consen S a emen : Pa ien consen was no applicable o his s udy.
Da a A ailabili y S a emen : Da a sha e upon easonable eques .
Acknowledgmen s:
The au ho s a e g a e ul o Guy Tempo al, Ch is ophe Bu el, and Sa ah Mi chel,
p o essionals in medical English, o hei assis ance in p oo eading he manusc ip . The au ho s
would like o hank he Kae Labs company o hei echnical suppo in lea ning how o use he
NIR-II imaging sys em du ing he expe imen a ion in he aining acili ies.
Con lic s o In e es :
Michele Diana is a membe o he Ad iso y Boa d o Diagnos ic G een. Michele
Diana and And ey Klymchenko a e he in en o s o he NICE echnology (Eu opean pa en ap-
plica ion No. 18305075.6). Jacques Ma escaux is he P esiden o IRCAD F ance, which is pa ly
unded by Ka l S o z and Med onic. Ma ía Ri a Rod íguez-Luna was inancially suppo ed by he
‘Ho izon 2020 esea ch and inno a ion p og am’ p ojec g an o he Eu opean Union unde he Ma ie
Skłodowska-Cu ie g an ag eemen No. 857894—CAST. The emaining au ho s ha e no con lic o
in e es o inancial ies o disclose.
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