T iden : A dual oxygena ion and
fluo escence imaging pla o m
o eal- ime and quan i a i e
su gical guidance
Sil è e Ségaud
1
, Luca Ba a elli
1
, E ic Felli
2
, Elisa Bannone
2
,
3
,
Lo enzo Cinelli
2
,
4
, Ma ía Ri a Rod íguez-Luna
1
,
2
,
Na iaki Okamo o
1
,
2
, Debo ah S Kelle
5
, Michel de Ma helin
1
,
Syl ain Lecle
1
,
6
, Michele Diana
1
,
2
and Syl ain Gioux
1
,
7
*
1
Uni e si y o S asbou g, ICube Labo a o y, Illki ch, F ance,
2
Resea ch Ins i u e Agains Diges i e
Cance (IRCAD), S asbou g, Alsace, F ance,
3
Depa men o Gene al and Panc ea ic Su ge y—The
Panc eas Ins i u e, Uni e si y o Ve ona, Ve ona, I aly,
4
Depa men o Gas oin es inal Su ge y, San
Ra aele Hospi al IRCCS, Milan, I aly,
5
Uni e si y o CA-Da is Medical Cen e , Depa men o Su ge y,
Sac amen o, CA, Uni ed S a es,
6
INSA S asbou g, S asbou g, Alsace, F ance,
7
In ui i e Su gical,
Aubonne, Swi ze land
Despi e ecen echnological p og ess in su gical guidance, cu en in aope a i e
assessmen o issue ha should be emo ed (e.g., cance ) o a oided (e.g., ne es)
is s ill pe o med subjec i ely. Op ical imaging is a non-con ac , non-in asi e
modali y ha has he po en ial o p o ide eedback ega ding he condi ion o
li ing issues by imaging ei he an exogenously adminis e ed con as agen o
endogenous cons i uen s such as hemoglobin, wa e , and lipids. As such, op ical
imaging is an a ac i e modali y o p o ide physiologically and s uc u ally ele an
in o ma ion o decision-making in eal- ime du ing su ge y. The T iden imaging
pla o m has been designed o eal- ime su gical guidance using s a e-o - he-a
op icalimaging.Thispla o miscapable o dual exogenous and endogenous
imaging owing o a unique fil e and sou ce combina ion, allowing o ake
ad an age o bo h imaging modali ies. This pla o m makes use o a eal- ime
and quan i a i e imaging me hod wo king in he spa ial equency domain, called
Single Snapsho imaging o Op ical P ope ies (SSOP). The T iden imaging pla o m
is designed o comply wi h all ele an s anda ds o clinical use. In his manusc ip ,
we fi s in oduce he a ionale o de eloping he T iden imaging pla o m. We
hen desc ibe fluo escence and endogenous imaging modali ies whe e we p esen
he de ails o he design, assess he pe o mance o he pla o m on he bench.
Finally, we pe o m he alida ion o he pla o m du ing an in i o p eclinical
expe imen . Al oge he , his wo k lays he ounda ion o ansla ing s a e-o - he-
a op ical imaging echnology o he clinic.
KEYWORDS
op ical imaging, oxygena ion imaging, fluo escence imaging, su gical guidance/
na iga ion, clinical ansla ion
OPEN ACCESS
EDITED BY
K is en M. Meibu ge ,
Poli ecnico di To ino, I aly
REVIEWED BY
Mengyang Liu,
Medical Uni e si y o Vienna, Aus ia
Dimi is Go pas,
Helmhol z Associa ion o Ge man
Resea ch Cen es (HZ), Ge many
*CORRESPONDENCE
Syl ain Gioux,
syl ain.gioux@in usu g.com
SPECIALTY SECTION
This a icle was submi ed o
Biopho onics,
a sec ion o he jou nal
F on ie s in Pho onics
RECEIVED 31 Augus 2022
ACCEPTED 18 Oc obe 2022
PUBLISHED 09 No embe 2022
CITATION
Ségaud S, Ba a elli L, Felli E, Bannone E,
Cinelli L, Rod íguez-Luna MR,
Okamo o N, Kelle DS, de Ma helin M,
Lecle S, Diana M and Gioux S (2022),
T iden : A dual oxygena ion and
fluo escence imaging pla o m o eal-
ime and quan i a i e su gical guidance.
F on . Pho onics 3:1032776.
doi: 10.3389/ pho .2022.1032776
COPYRIGHT
© 2022 Ségaud, Ba a elli, Felli, Bannone,
Cinelli, Rod íguez-Luna, Okamo o,
Kelle , de Ma helin, Lecle , Diana and
Gioux. This is an open-access a icle
dis ibu ed unde he e ms o he
C ea i e Commons A ibu ion License
(CC BY). The use, dis ibu ion o
ep oduc ion in o he o ums is
pe mi ed, p o ided he o iginal
au ho (s) and he copy igh owne (s) a e
c edi ed and ha he o iginal
publica ion in his jou nal is ci ed, in
acco dance wi h accep ed academic
p ac ice. No use, dis ibu ion o
ep oduc ion is pe mi ed which does
no comply wi h hese e ms.
F on ie s in Pho onics on ie sin.o g01
TYPE O iginal Resea ch
PUBLISHED 09 No embe 2022
DOI 10.3389/ pho .2022.1032776
1 In oduc ion
The capabili ies o nea -in a ed (NIR) op ical imaging
g ea ly expanded o e he pas ew yea s, p o iding eedback
o issue s a us assessmen du ing su ge y, and pa icula ly
blood pe usion assessmen . In he con ex o his wo k,
se e al in aope a i e op ical imaging echnologies allowing
non-con ac , la ge field-o - iew mac oscopic (i.e., g ea e han
100 cm
2
) imaging o he su gical field ha e been de eloped all he
way om concep o clinical ials es ing. These echnologies
di e om mic oscopic imaging echnologies ha ing smalle
fields o iew and equi ing he ins umen o be in con ac o
e y close o he specimen. In his con ex , he de elopmen o
fluo escence imaging o su gical guidance pa ed he way wi h
he g owing numbe o a ailable con as agen s and he
de elopmen o new mac oscopic imaging echnologies (Gioux
e al., 2010;Gibbs, 2012;Vah meije e al., 2013). Howe e , he
lack o quan i a i e capabili ies leads o subjec i e assessmen o
fluo escence images, in oducing a iabili y in he su gical
ou come (Pogue e al., 2018a;Pogue e al., 2018b;Mieog
e al., 2022). Al e na i ely, he abili y o mul ispec al and
hype spec al endogenous imaging o ex ac unc ional
pa ame e s o clinical applica ions has been epo ed o
su gical guidance (Lu and Fei, 2014;Shapey e al., 2019;
Clancy e al., 2020). Ye , i su e s om he lack o
quan i a i e ex ac ion o issue cons i uen s’concen a ions
and canno p o ide eal- ime eedback in aope a i ely.
Spa ial F equency Domain Imaging (SFDI) is a ela i ely
ecen op ical imaging me hod ha has he capabili y o pe o m
quan i a i e imaging o hemoglobin concen a ions, and in u n
o issue oxygena ion o e la ge fields o iew (>100 cm
2
)
(Dogni z and Wagnie es, 1998;Cuccia e al., 2009;Gioux
e al., 2019). Recen de elopmen s in acquisi ion and
p ocessing led o a eal- ime implemen a ion o SFDI–namely
Single Snapsho imaging o Op ical P ope ies (SSOP)
(Ve andie and Gioux, 2013; an de Giessen e al., 2015).
The mos ad anced o m o SSOP makes use o deep
lea ning-based p ocessing, leading o high-quali y images
while p ese ing he eal- ime quan i a i e capabili ies o he
me hod (Aguenounon e al., 2020;Smi h e al., 2022).
To o e come he lack o ools capable o quan i a i e
endogenous imaging o issue assessmen in clinical se ings,
we p opose a no el imaging pla o m, called T iden , in eg a ing
he la es SSOP de elopmen s o pe o ming eal- ime
oxygena ion imaging as well as fluo escence imaging.
Combining hese s a e-o - he-a imaging modali ies in a
single imaging pla o m enables he compa ison o hei
espec i e pe o mances as well as he po en ial o hei
combina ion.
In his manusc ip , ollowing a backg ound sec ion on op ical
imaging o su ge y, he T iden imaging pla o m is desc ibed in
de ail, as well as he fluo escence and oxygena ion imaging
me hods used. Nex , bench cha ac e iza ion expe imen s a e
p esen ed, showing he pe o mances o he pla o m o
op ical p ope ies imaging and fluo escence de ec ion. Finally,
in i o alida ion demons a es he abili y o he imaging
pla o m o dynamically de ec ischemia in he small bowel by
measu ing issue oxygena ion and fluo escence in eal- ime.
2 Backg ound
2.1 Quan i a i e op ical imaging o
su ge y
Se e al implemen a ions o ei he fluo escence imaging o
oxygena ion wide-field (>100 cm
2
)imagingexis in he
li e a u e (DSouza e al., 2016;Shapey e al., 2019;Clancy
e al., 2020). Depending on he imaging me hodology used,
hese implemen a ions g ea ly a y in hei capabili y o
quan i y he measu ed signal. Wi hou ying o p o ide an
ex ensi e e iew, i is wo h men ioning ha he mos
common o m o op ical imaging o ei he fluo escence o
oxygena ion consis s o using empo ally and spa ially
cons an illumina ions (Bigio and Fan ini, 2016). These
me hods a e inhe en ly limi ed in hei capaci y o quan i y
signalssince heya eunable osepa a esca e ing om
abso p ion, c ea ing a c oss alk be ween he wo sou ces o
con as . By con as , me hods using modula ions in ei he
ime o space allow o he sepa a ion o sca e ing and
abso p ion, he eby allowing a mo e accu a e quan ifica ion
o he signals.
In his wo k, we use a me hod called Spa ial F equency
Domain Imaging (SFDI). A ecen e iew has been published
in he li e a u e, explaining in de ail he p inciples, acquisi ion
and p ocessing me hods as well as applica ions (Gioux e al.,
2019). In a nu shell, SFDI elies on he p ojec ion o a spa ially
modula ed illumina ion pa e n (also called s uc u ed
illumina ion, ypically a sinusoidally modula ed pa e n) and
he quan ifica ion o he ampli ude dampening o he
eflec ance pa e n as a unc ion o spa ial equency. A ligh
p opaga ion model is hen used ( ypically Mon e Ca lo) o ela e
he dampening o he ampli ude o he eflec ance pa e n o he
op ical p ope ies o he medium (abso p ion coe ficien and
educed sca e ing coe ficien ). Ou g oup de eloped a eal- ime
implemen a ion o SFDI, called Single Snapsho o Op ical
P ope ies (SSOP) capable o cap u ing abso p ion and
educed sca e ing coe ficien s as well as p ofile in o ma ion
( o 3D p ofile co ec ion) o a sample wi h only one image
(Ve andie and Gioux, 2013; an de Giessen e al., 2015;Angelo
e al., 2017). The la es implemen a ion makes use o GPU, as
lookup ables and deep lea ning o allow accu a e and high
image-quali y imaging wi h p ofile co ec ion in eal- ime
(Angelo e al., 2016;Panig ahi and Gioux, 2018;Schmid
e al., 2019;Aguenounon e al., 2020). Resou ces ega ding he
ins umen al and p ocessing aspec s o SFDI and SSOP,
F on ie s in Pho onics on ie sin.o g02
Ségaud e al. 10.3389/ pho .2022.1032776
including ma e ial e e ences and p ocessing code, a e eely
a ailable in OpenSFDI (Applega e e al., 2020).
2.2 Fluo escence imaging
The mos common o m o fluo escence imaging o image-
guided su ge y elies on he injec ion o an exogenous con as
agen o highligh a pa icula s uc u e (e.g., lymph node, essels,
umo ) (F angioni, 2003;N ziach is os, 2006;Gioux e al., 2010;
Gibbs, 2012;Vah meije e al., 2013;Mieog e al., 2022). A
dedica ed imaging sys em ha ma ches he op ical p ope ies
o he con as agen (namely abso p ion and emission spec a) is
hen used o highligh he p esence o he con as agen in eal-
ime du ing he su ge y. Cu en implemen a ions used in he
clinic (many being comme cial p oduc s) a e quali a i e in
na u e, meaning he fluo escence signal measu ed by he
imaging sys em is no di ec ly ela ed o he local amoun o
con as agen (i.e., i s concen a ion). The fluo escence signal is
dependen on mul iple ex e nal and in e nal ac o s such as he
illumina ion homogenei y, he dis ance be ween he imaging
sys em and he su gical field, he local issue op ical p ope ies
(abso p ion and sca e ing p ope ies), he a ia ion o he
fluo escence p ope ies due o he dye en i onmen , o he
quan um yield o he molecule. Recen e o has ied o
add ess hese challenges (Pogue e al., 2018a;Pogue e al.,
2018b;Mieog e al., 2022). Some cu en comme cial sys ems
allow o co ec o he dis ance be ween he imaging sys em and
he su gical field, and some e en co ec o he illumina ion
inhomogenei ies. Resea ch p o o ypes ha e been designed o
u he co ec no only dis ance and field homogenei y, bu
also op ical p ope ies, e en in eal- ime. Finally, he empo al
beha io o he fluo escence signal is being in es iga ed as a
sou ce o con as , no ably o he imaging o blood pe usion
(Ma sui e al., 2009;Diana e al., 2015;Meije e al., 2021). This
me hod e e ed o as fluo escence dynamics is less dependen on
se e al ex e nal and in e nal ac o s.
In he implemen a ion desc ibed in his wo k, we chose o
enable h ee modes o fluo escence imaging in he same
p o o ype. The fi s mode consis s o illumina ing he field
wi h a empo ally cons an (called con inuous wa e, CW)
nea in a ed illumina ion. CW fluo escence imaging is he
mos common implemen a ion bo h comme cially and in
esea ch (F angioni, 2003;Gioux e al., 2010). The second
mode consis s o cap u ing bo h he fluo escence image and
he exci a ion illumina ion. One hen di ides he fluo escence
image by he exci a ion image o au oma ically co ec o
dis ance, illumina ion inhomogenei y and pa ially o op ical
p ope ies (N ziach is os e al., 2005;Themelis e al., 2009). The
hi d mode consis s o using SFDI o SSOP o ex ac he op ical
p ope ies o he su gical field a bo h he exci a ion and emission
wa eleng h and use his in o ma ion o co ec o dis ance,
sample p ofile, illumina ion inhomogenei y and op ical
p ope ies. The me hod used o ob ain he so-called
quan i a i e fluo escence images ha e been p e iously
desc ibed in he li e a u e (Sibai e al., 2019;Valdes e al.,
2019). We use ou own me hod called qF-SSOP (Valdes e al.,
2017). No e ha o fluo escence imaging, he exci a ion
wa eleng h and fil e used o de ec he signal a e dependen
on he con as agen imaged. In ou case, we in end o image
indocyanine g een (ICG), and om p io wo k, we use an
exci a ion a 760 nm and collec ion h ough fil e s s a ing a
780 nm. Mo e de ails abou ou fil a ion s a egy a e desc ibed
in sec ion 2.4.2.
2.3 Oxygena ion imaging
Oxygena ion imaging has gained inc easing in e es o
su gical applica ions (DSouza e al., 2016;Kohle e al., 2019;
Shapey e al., 2019;Clancy e al., 2020;Felli e al., 2020;Felli e al.,
2021). Oxygen sa u a ion is compu ed as he a io o he
concen a ion o oxy-hemoglobin o he concen a ion o o al
hemoglobin (oxy-hemoglobin + deoxy-hemoglobin). No e ha
oxygen sa u a ion he e e e s o a mix o a e ial and enous
blood, as opposed o pulse oxime y ha only measu es he
amoun o oxygen sa u a ion in a e ial blood. Mos me hods
used o oxygena ion imaging ely on eflec ance imaging a
se e al wa eleng hs, also called mul ispec al imaging, o
hype spec al imaging in cases whe e a la ge numbe o
wa eleng hs a e used (e.g., >10 wa eleng hs). These me hods
ely on CW illumina ion and ypically fi he measu ed
eflec ance spec um o a heo e ical spec um and ex ac an
oxygena ion alue. In he case o oxygena ion imaging, because i
is a a io (oxy-hemoglobin o o al hemoglobin), he influence o
sca e ing and dis ance be ween he sample and he imaging
sys em can be managed. Se e al au ho s demons a e he use o
hese CW mul ispec al and hype spec al me hods in su ge y.
Ano he app oach o oxygena ion imaging consis s o
p ope ly sepa a ing sca e ing and abso p ion on he
measu ed signal by using a quan i a i e op ical imaging
me hod, such as SFDI (Gioux e al., 2011;Pon ico o e al.,
2013). In his case, he abso p ion coe ficien a a minimum o
wo wa eleng hs is hen used in conjunc ion wi h Bee ’s law o
di ec ly quan i y he concen a ion o oxy-hemoglobin and
deoxy-hemoglobin. These alues a e hen used o compu e he
oxygen sa u a ion o he sample. In ou case, p io wo k in SFDI
de e mined ha op imal wa eleng hs o oxygena ion imaging
we e close o 665 nm and 860 nm (Mazha e al., 2010). Ou
g oup and o he s ha e ansla ed simila echnology o
p eclinical and clinical expe imen s (Gioux e al., 2011;
Nadeau e al., 2013;Pon ico o e al., 2013;Ghijsen e al.,
2018;Schmid e al., 2019;Weinkau e al., 2019;Chen and
Du , 2020;Ren e al., 2020;Zhao e al., 2021;Lyu e al., 2022).
Howe e , none o he p io wo k includes a eal- ime
me hodology and combines he measu emen o oxygena ion
F on ie s in Pho onics on ie sin.o g03
Ségaud e al. 10.3389/ pho .2022.1032776
wi h he measu emen o fluo escence. The T iden imaging
pla o m enables such ea u e o unde s and po en ial clinical
use o ei he o bo h echnologies.
3 Ma e ials and me hods
3.1 Sys em design
This sec ion desc ibes he design o he imaging pla o m o
each subsys em. An o e iew o he comple e sys em is gi en in
Figu e 1A. This medical ca -based pla o m ea u es a h ee
channel-imaging head, fibe -coupled whi e ligh sou ces and
lase sou ces, and a wo ks a ion.
3.1.1 Ca and a m
The pla o m is buil upon a cus om medical-g ade ca
(Symbio ca , ITD, Neubibe g, Ge many). The wheeled base
is equipped wi h an isola ion ans o me o egula e powe
supply o he de ice and coun e weigh s o sa ely balance he
assembly (complian wi h IEC60601). Two a icula ed a ms
bea display sc eens bo h o he su geons and he ope a o .
A hi d a icula ed a m (Eli e 5220, ICW, Med o d, OR,
Uni ed S a es) is fixed o he cen al column o
accommoda e he imaging head. I s la ge each enables a
use o ope a e he sys em while keeping dis ance om he
s e ile field. Cables can be guided om he de ices h ough
he cen al column and cable e aine on he a icula ed a m.
Powe is supplied o he imaging head using a 24 V AC/DC
con e e (AKM90PS24, XP Powe , Singapo e). All h ee
a icula ed a ms con enien ly old o s o age. The wide
cas o s and la ge handle p o ide good mobili y when
deploying o olling ou he pla o m.
3.1.2 Imaging head and fil e s design
The imaging head is depic ed in Figu es 1B,C. The
op omechanical design ea u es h ee independen imaging
channels: one isible ligh channel (COLOR) and wo NIR
channels (NIR1 and NIR2). These channels a e co- egis e ed
using il ing mi o s, p oducing a field o iew o 12 cm × 15 cm.
The de ec ion op ics enable fine ocusing and indi idual ape u e
con ol. An RGB came a (GO-5000C-USB, JAI L d., Kanagawa,
Japan) and wo monoch ome came as (GO-5000M-USB, JAI
L d., Kanagawa, Japan; Edge 4.2, Exceli as PCO GmbH, Kelheim,
FIGURE 1
T iden imaging pla o m: (A) Pic u e o he en i e imaging pla o m, including he medical-g ade a , he a icula ed a m, he imaging head, he
ligh sou ces (whi e ligh and lase s), he compu e and he displays; (B) pic u e o he imaging head a he end o he a icula ed a m including he
came as, he Dp omechanical coupling sys em, he p ojec o and he op ical lenses; (C) de ailed schema ics o he imaging head including he
came as, he op omechanical coupling sys em, he op ical lenses, he dich oic mi o s, he ligh sou ces, he illumina ion ing, he p ojec o and
he ligh pa hs; (D) de ails o he fil e s design o he imaging sys em: no e he sepa a ion be ween he colo , NIR1 and NIR2 came as in combina ion
wi h he ligh sou ces allowing he imaging o he su gical field in colo , oxygena ion o fluo escence.
F on ie s in Pho onics on ie sin.o g04
Ségaud e al. 10.3389/ pho .2022.1032776
Ge many) a e used o he de ec ion in he COLOR, NIR1 and
NIR2 channels, espec i ely.
Cus om fil e se s (Ch oma Inc., Bellows Falls, VT, Uni ed S a es)
p o ide wa ebands selec ion o op ical p ope ies and fluo escence
imaging wi h minimized c oss alk be ween channels. Two fil e cubes
(Axio cubes, Zeiss, Obe kochen, Ge many) a e composed o wo
emission fil e s and a dich oic mi o . These fil e s can be swapped
easily wi h minimal e-alignmen depending on he desi ed imaging
configu a ion. Figu e 1D depic s he fil e design in combina ion wi h
he sou ces o he p esen ed configu a ion o oxygena ion imaging
and 800 nm fluo escence imaging. A fi s fil e cube allows o he
isola ion o he COLOR channel (400–650 nm). A second fil e cube
sepa a es he NIR1 and NIR2 channels. The NIR1 channel in he
p esen ed configu a ion allows o he de ec ion o ei he he 665 nm
o oxygena ion imaging o 760 nm o fluo escence imaging. The
NIR2 channel in he p esen ed configu a ion allows o he de ec ion
o ei he he emission fluo escence signal om ICG abo e 780 nm o
860 nm o oxygena ion imaging. No e ha he dich oic fil e s op ical
densi y is a ound 2 and he channels emission fil e s op ical densi ies
a e a ound 6–7. This configu a ion allows app op ia e sepa a ion o
he isible ana omical in o ma ion in he COLOR channel,
concu en ly wi h ei he oxygena ion imaging o fluo escence in
NIR1 and NIR2 channels.
3.1.3 Whi e ligh sou ce
Ligh emi ing diodes (LEDs) based lamps (LO-35,
Fibe op ics Technologies Inc., Pom e , CT, Uni ed S a es)
wi h cus om 650 nm sho -pass fil e s and fibe bundles
p o ide a b igh illumina ion o he su gical field. Though he
LEDs emi low amoun s o in a ed ligh , he addi ional fil e
ensu es an op imal isible illumina ion while p ese ing he NIR
channels om backg ound noise. The wo fibe bundles a e spli
in wo ou pu s each o p oduce a mul i-angle illumina ion om
he imaging head o e a la ge a ea o e 50 cm diame e a a 45 cm
wo king dis ance. An illumina ion ing wi h cus om lensing
allows o illumina e he field in he mos homogenous manne .
3.1.4 Lase sou ce
Cus om lase sou ces we e designed o equip he pla o m. A
fi s sou ce is dedica ed o he p ojec o illumina ion o collec
eflec ance da a o op ical p ope ies imaging and a second
sou ce is dedica ed o fluo escence imaging. The wo sou ces a e
e e ed o as eflec ance sou ce and fluo escence sou ce,
espec i ely o hei pu pose. Figu es 2A,B p esen s one
comple e sou ce closed and open ( op- iew) espec i ely.
A single sou ce houses six independen modules ha can be
loaded wi h di e en lase diodes (LDX Op onics, Ma y ille,
TN, Uni ed S a es) o achie e mul ispec al imaging, o o ma ch
he exci a ion o fluo opho es o in e es . Iden ical lase diodes
can also be combined o p o ide highe illumina ion powe . Fo
eal- ime oxygena ion imaging, a combina ion o ou 665 nm
(2 W each) and wo 860 nm (5 W each) lase diodes equip he
eflec ance sou ce. In o de o exci e 800 nm
fluo opho es–especially indocyanine g een (ICG)—six 760 nm
(6 W each) lase diodes a e moun ed in he fluo escence sou ce.
Each diode is he mally egula ed by a he moelec ic coole
(TEC) (TEC-15.4-3.9-33.4-72-40-CH4,7, A c ic TEC
Technologies, Do mund, Ge many) and a he mis o placed
on he lase diode chassis (TH10K, Tho labs, New on, NJ,
Uni ed S a es). Indi idual TEC d i e s (PTC5K-CH,
Wa eleng h elec onics, Bozeman, MT, Uni ed S a es) enable
empe a u e egula ion and moni o ing o he diodes. Simila ly,
lase diode d i e s (LD10CHA, wa eleng h elec onics,
Bozeman, MT, Uni ed S a es) con ol he lase ou pu powe ,
and moni o ing o he in eg a ed pho odiode eedback. A cus om
op omechanical assembly ea u ing op ical fil e s (Ch oma Inc.,
Bellows Falls, VT, Uni ed S a es) and g aded-index lenses
(Newpo , I ine, CA, Uni ed S a es) pe o ms he lase
ou pu coupling in o a dedica ed fibe bundle (Ce amOp ec,
Bonn, Ge many). In he case o he eflec ance sou ce, he
modules a e coupled in o a 6-legged fibe bundle combined in
a single ou pu o coupling in he p ojec o . In he fluo escence
sou ce, he 6 legs o he bundle con ain mul iple fibe s which a e
andomized and edis ibu ed in o 6 legs ha a e plugged in an
illumina ion ing. In addi ion, each module is in e aced wi h
USB analog inpu /oupu modules (USB-202, Measu emen
Compu ing, No on, MA, Uni ed S a es) and cus om
elec onics o con ol he modules and eco d eedback signals
om a compu e . The in e acing o a single module is depic ed
in Figu e 2C.
As shown in Figu e 2D, in o de o dissipa e he g ea
amoun s o hea gene a ed by he lase diode cooling and
di e en d i e s, all he hea ing modules componen s a e
moun ed on a unnel hea sink equipped will push-pull ans.
This design p oduces a compac assembly and while limi ing
noise due o he limi ed numbe o ans and hei la ge diame e .
3.1.5 Illumina ion
Two independen illumina ions can be used wi h he T iden
imaging pla o m. The fi s illumina ion is a single illumina ion
ing con aining ou lensed ou pu s o he whi e ligh ( om he
LED whi e ligh sou ce), and six SMA ou pu s o he CW
fluo escence illumina ion ( om he fluo escence lase diode
sou ce). The pa e n and lensing ha e been op imized o he
bes o ou e o o p o ide he mos homogeneous illumina ion
possible.
A p ojec o based on a digi al mic omi o de ice
equipped wi h a NIR compa ible p ojec ion op ics
(V7000 HiSpeed and STAR-CORE, Vialux GmbH,
Chemni z, Ge many) and a cus om fibe coupling
adap e enables he p ojec ion o pa e ns a a 45 cm
wo king dis ance o op ical p ope ies imaging using
SFDI. Linea pola ize s (RCV8N2EC, Mox ek, O em,
UT, Uni ed S a es) a e c oss-pola ized in on o he
p ojec ion and de ec ion pa hs o ejec specula
eflec ions. One 5 V DC/DC con e e (PYB30-Q24-S5-
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U, CUI Inc., Tuala in, OR, Uni ed S a es) p o ides powe
supply o he digi al mic omi o de ice (DMD) boa d.
3.1.6 Wo ks a ion
A high-pe o mance wo ks a ion has been assembled o
achie e he imaging de ice con ol, da a p ocessing, da a
s o age managemen and isualiza ion. A Came aLink HS
ame g abbe (ME5 Ma a hon AF2, Silicon So wa e,
Mannheim, Ge many) and an addi ional USB3.0 con olle
ensu e he communica ion wi h he imaging head. Powe ul
CPU (In el Co e i5-10600K, In el, San a Cla a, CA,
Uni ed S a es) and GPU (Quad o RTX 4000, NVIDIA, San a
Cla a, CA, Uni ed S a es) and 64 GB o high-speed RAM can
pe o m in ensi e da a p ocessing and isualiza ion on mul iple
displays. Finally, 5 TB o as SSD s o age a e ins alled o da a
s eam managemen up o 1 GB/s.
3.2 Sys em es ing
3.2.1 Op ical pe o mances
A USAF1951 esolu ion cha (RES-2, Newpo , I ine, CA,
Uni ed S a es) was placed unde he imaging head a 45 cm
wo king dis ance and imaged wi h each channel: COLOR,
NIR1 and NIR2. Op ical p ope ies ex ac ion was cha ac e ized
by imaging an a ay o 12 issue-mimicking phan oms o 6 × 6 ×
2.5 cm. These cu ed silicone-base Polydime hylsiloxane (PDMS)
samples we e ab ica ed using a ying amoun s o alcohol-soluble
nig osine and Ti anium dioxide (TiO
2
) o une he abso p ion and
sca e ing o he ma e ial, espec i ely, spanning a la ge ange o
op ical p ope ies o be measu ed: µ
a
anging om 0.013 o
0.08 mm
−1
and µ
s
’ anging om 0.75 o 1.8 mm
−1
a 665 nm.
These samples we e imaged a 665 nm and 860 nm unde a
0.2 mm
−1
spa ial equency illumina ion and p ocessed using
he deep lea ning-based implemen a ion o SSOP. The
measu emen s we e compa ed o hose ob ained wi h a
e e ence bench op imaging sys em using 7 phase-SFDI. Tha
e e ence SFDI sys em was alida ed in a mul i-labo a o y
pe o mance assessmen o di use op ics ins umen s (Bi Map
exe cise) (Lanka e al., 2022) and used o ob ain he e e ence
op ical p ope y alues o his wo k.
3.2.2 Illumina ion
Peak fluence a es we e measu ed a 45 cm wo king dis ance
in he cen e o he field o iew using a digi al handheld powe
me e (PM100D, Tho labs, New on, NJ, Uni ed S a es) a 665 nm
and 860 nm while p ojec ing a con inuous wa e pa e n, and a
760 nm by fibe illumina ion.
FIGURE 2
Lase ligh sou ce: (A) Pic u e o he ligh sou ce closed; (B) pic u e o he ligh sou ce opened ( op iew); (C) de ailed schema ics o a single lase
diode uni including he lase diode (LD) and i s d i e , he he moelec ic coole (TEC) and i s d i e , he he mis o , he op omechanical module and
he ou pu fibe ; (D) de ails o he cooling design o he sou ce: no e ha he single lase diode uni s (D1 o D6) a e assembled on o a unique hea sink
wi h o ced ai flow (in and ou ) o mo e e ficien cooling while main aining a compac design.
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3.2.3 Sensi i i y and noise
The sensi i i y o he NIR2 channel o fluo escence
de ec ion was assessed by imaging eigh di e en dilu ions o
ICG dilu ed in dime hyl sul oxide (DMSO) a concen a ions
anging om 25 nM up o 800 nM. The ials we e placed a
45 cm om he imaging head and imaged wi h a ying exposu e
se ings successi ely on o wo di e en backg ound ma e ials: a
la ge issue-mimicking phan om wi h µ
a
= 0.012 mm
−1
and µ
s
’=
1.08 mm
−1
a 665 nm and a shee o whi e pape . A e age
fluo escence signals we e ex ac ed and compa ed o he noise
floo le el o sensi i i y assessmen . Sensi i i y was defined as
he concen a ion p oducing an in ensi y wice as high as he
noise floo le el.
In o de o quan i y he backg ound noise in he fluo escence
images, he con ibu ions om da k noise and bo h whi e ligh
and exci a ion ligh leakage we e measu ed. Images o he
p e iously men ioned backg ound ma e ials om he
NIR2 channel we e acqui ed wi h a ying exposu e
se ings–exposu e ime, senso binning–wi h successi ely all
ligh s swi ched o , and whi e ligh and exci a ion lase ligh
swi ched on.
3.3 P eclinical expe imen s
3.3.1 Bowel ischemia model
One la ge swine was in ol ed in his non-su i al s udy,
which ecei ed ull app o al om he local E hical Commi ee on
Animal Expe imen a ion (ICOMETH No. 038.2019.01.121) and
by he F ench Minis y o Supe io Educa ion and Resea ch
(MESR) unde he ollowing e e ence: APAFIS #20819-
2019052411591088 3. A e pe o ming a midline
lapa o omy, a 20 cm small bowel loop was exposed. A cen al
egion was delinea ed, and co esponding a cade b anches we e
isola ed. A clamp was used o c ea e an occlusion on hese essels,
causing ischemia on a limi ed po ion o he bowel while he es
o he loop emained pe used. The occlusion was main ained
du ing 14 min un il elease.
3.3.2 Fluo escence imaging
A dose o 4 mg o ICG powde dilu ed in 1.6 ml o 5% glucose
solu ion (In acyanine, SERB, Pa is, F ance) was injec ed
in a enously 2 min be o e occlusion elease. Colo images
om he COLOR channel and fluo escence images om he
NIR2 channel we e acqui ed s a ing om he injec ion
imepoin and o 2 min and 30 s. The imaging wo king
dis ance was main ained a 45 cm. Fluo escence exci a ion was
p oduced using 760 nm lase ligh .
3.3.3 Oxygena ion imaging
A sinusoidal pa e n wi h spa ial equency o 0.2 mm
−1
was
p ojec ed a bo h 665 nm and 860 nm wa eleng hs using he
DMD p ojec o . Reflec ance images om he NIR1 and
NIR2 channels we e acqui ed, as well as colo images om
he COLOR channel o ana omical e e ence. Images we e
eco ded s a ing 2 min be o e occlusion o es ablish he
oxygen sa u a ion baseline, and du ing 12 min a e occlusion.
A e pe o ming fluo escence imaging, oxygena ion imaging
esumed o eco d o 3 min du ing he end o he epe usion
phase.
4 Resul s
4.1 Sys em es ing
4.1.1 Op ical pe o mances
A 45 cm wo king dis ance and pe o ming 2 × 2 binning on
he came a senso ( o inc eased sensi i i y), he op ical sys em
p oduces images o a 12 × 15 cm field-o - iew, wi h a esolu ion
o 1024 × 1280 pixels. The measu ed la e al esolu ion wi h ully
open ape u es o channels COLOR and NIR2 was 5.04 lp/mm
and 5.66 lp/mm o he NIR1 channel.
A se o 12 issue-mimicking phan oms was imaged fi s wi h
a bench op imaging sys em using 7 phase-SFDI as e e ence. The
images p oduced by he clinical pla o m we e p ocessed using
deep lea ning-based SSOP. High image quali y and good
accu acy wi h espec o op ical p ope ies ex ac ion we e
achie ed. Figu e 3 p esen s he ex ac ed op ical p ope ies
maps and e o es ima ion in compa ison o s anda d SFDI.
E o s in µ
a
and µ
s
’measu emen s we e espec i ely below 2.7%
and 2.1% o bo h 665 nm and 860 nm oxygena ion imaging
wa eleng hs.
4.1.2 Illumina ion
A con inuous-wa e illumina ion was p oduced using he
DMD p ojec o a bo h 665 nm and 860 nm oxygena ion
imaging wa eleng hs. The p ojec o has a small Gaussian-like
a ia ion o in ensi y ac oss he p ojec ed field (55% maximum).
This inhomogenei y does no a ec he pe o mance o he
sys em as he signal- o-noise a io allows p ope p ocessing o
he da a a all loca ions o he image. A 45 cm wo king dis ance,
a powe me e p obe was placed in he cen e o he field-o - iew.
Fluence a es o 0.93 mW/cm
2
a 665 nm and 1.51 mW/cm
2
a
860 nm we e measu ed and classified as lase Class 1 as pe
IEC60825. The fluo escence exci a ion field a 760 nm showed a
peak fluence a e o 31.51 mW/cm
2
and classified as lase Class
3 R as pe IEC60825.
4.1.3 Sensi i i y and noise
A se o 8 ICG dilu ions wi h concen a ions anging om
25 nM o 800 nM was imaged in he same p e iously s a ed
condi ions. Figu e 4A shows he images om he COLOR and
NIR2 channels as well as an o e lay. Figu e 4B shows he
e olu ion o in ensi y in he NIR2 images o each dilu ion a
a ious exposu e imes wi h fixed 2 × 2 senso binning as well as
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using a silicone phan om as backg ound. The in ensi y inc eases
linea ly wi h he exposu e ime un il eaching sa u a ion. F om
he en i e dilu ion se ies, he sensi i i y was es ima ed by
measu ing he concen a ion co esponding o an in ensi y
2 imes highe han he noise floo le el, yielding a signal- o-
backg ound a io o 2. A 40 ms exposu e ime and 2 × 2 binning
ac o , he sensi i i y was es ima ed a 18.8 nM.
Backg ound noise in he NIR2 channel was measu ed
using successi ely a issue-mimicking phan om and a shee o
whi e pape placed a 45 cm wo king dis ance. Figu e 4C
shows hee olu iono da knoise and leakage wi h exposu e
se ings and backg ound ma e ials. Da k noise was fi s
measu ed wi h exposu e imes anging om 5 ms o
160 ms and senso binning ac o s o 1 × 1, 2 × 2, and 4 ×
4. Noise le els ep esen ing 0.1%, 0.6%, and 2.4% o he
dynamic ange o he NIR2 came a we e measu ed
espec i ely wi h 1 × 1, 2 × 2, and 4 × 4 binning ac o s.
The same se o images was acqui ed wi h ully open ape u e,
and bo h whi e ligh and 760 nm lase exci a ion ligh
swi ched on. Noise le els we e on a e age 17% and 46%
highe han he da k noise using a phan om backg ound
o a pape backg ound, co esponding mos ly o exci a ion
ligh leakage in o he NIR2 channel.
4.2 P eclinical expe imen s
Figu es 5A,B shows he esul s o he p eclinical expe imen s
in COLOR, fluo escence and oxygena ion. A compa ison o
fluo escence and oxygena ion imaging in gi en a h ee
imepoin s: du ing he baseline phase, a e 12 min o
occlusion and 1 min a e epe usion.
4.2.1 Fluo escence imaging
A 12 min in o he occlusion phase, a dose o ICG was
in a enously injec ed. Images om he NIR2 and COLOR
channels we e eco ded a 12 ames pe second wi h 50 ms
exposu e ime and 2 × 2 senso binning while illumina ing he
su gical field wi h 760 nm lase ligh . Fluo escence in ensi y was
moni o ed in a 120 × 180 pixels egion o in e es (ROI) loca ed
in he ischemic a ea and in a 50 × 50 pixels ROI loca ed in a
pe used a ea. The fluo escence signal eached i s peak in he
pe used a ea 35 s a e injec ion be o e dec easing and
s abilizing a 58% o he peak in ensi y 1 min la e .
A e 14 min o occlusion, he clamp was eleased. The
fluo escence in ensi y immedia ely inc eases in he ischemic
a ea, as he pe usion esumes. When s abilizing, he in ensi y
eaches 72% o he in ensi y in he con ol a ea.
FIGURE 3
Bench esul s: endogenous imaging: (A) Tissue mimicking phan oms ha ing di e en abso p ion and educed sca e ing p ope ies imaged
wi h he T iden imaging pla o m: op ical p ope ies a e imaged on all phan oms using he NIR1 (665 nm) and NIR2 (860 nm) came as; (B) plo s
compa ing he esul s ob ained using a e e encing SFDI acquisi ion me hod on a bench sys em compa ed wi h he esul s ob ained wi h he SSOP
acquisi ion me hod on he T iden pla o m: no e he good ag eemen in abso p ion and educed sca e ing (e o s lowe han 2.7% and 2.1%,
espec i ely).
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4.2.2 Oxygena ion imaging
Oxygena ion imaging was achie ed a 12 ames pe second
using s uc u ed illumina ion a 0.2 mm
−1
spa ial equency a
665 nm and 860 nm. Figu e 5B shows he oxygen sa u a ion
ends o he wo ROIs o e ime. Du ing he baseline phase,
bo h ROIs show s able S O
2
a es o 77.9% and 74.6% espec i ely
in he con ol and ischemic a eas. While he pe used a ea
emains 4% o i s baseline alue o S O
2
, oxygena ion in he
ischemic a ea dec eases un il eaching 63.8% a e 12 min o
occlusion. F om 2 min o 30 s a e he occlusion elease, he S O
2
a e dec eases back i s baseline s a e, eaching 75% a e 5 min
and 30 s o epe usion.
4.2.3 Video
A ideo is included in he supplemen a y ma e ial. I p esen s he
me ged o he oxygena ion and fluo escence esul s o e he cou se o
he expe imen . In i o oxygena ion and fluo escence imaging. An
occlusion o a po ion o a bowel loop was pe o med o 14 min.
Oxygena ion was moni o ed p io o clamping and du ing he 12 fi s
minu es o occlusion. Fluo escence images we e eco ded om he ICG
in a enous injec ion un il 30 s a e elease. Oxygena ion imaging
esumed o he 3 min o he epe usion phase. The absence o
fluo escence signal in he ischemic a ea co ela es wi h he low
S O2 a es obse ed. A e epe usion, his a ea shows fluo escence
signal le els and S O2 a es close o hoseo hepe useda eas.
Oxygena ion a es a e s eady in he con ol a ea whe eas ischemia
could be de ec ed and quan ified in he occluded a ea.
5 Discussion
We p esen ed he design and es ing o a clinically-
compa ible imaging pla o m o eal- ime quan i a i e op ical
imaging du ing su ge y. The pla o m is designed o image ei he
fluo escence in h ee modes (CW, semi-quan i a i e and
quan i a i e) o oxygena ion. The pla o m imaging modali y
can be changed on he fly du ing he su ge y allowing o compa e
bo h fluo escence and oxygena ion modali ies.
The bench cha ac e iza ion o he pla o m demons a es i s
pe o mances o op ical p ope ies imaging and fluo escence
imaging. Fi s , he compa ison be ween issue-mimicking
phan om op ical p ope ies ob ained om s anda d p ofile
co ec ed SFDI and s a e-o - he-a deep lea ning-based
p ofile co ec ed SSOP shows good ag eemen o bo h
abso p ion and educed sca e ing, a 665 nm and 860 nm, as
expec ed om p e ious published alida ion o SSOP. A e age
e o below 2.7% in abso p ion and 2.1% in educed sca e ing
we e obse ed. The conside ed op ical p ope ies we e ma ching
FIGURE 4
Bench esul s: fluo escence imaging: (A) Resul s ob ained wi h he T iden imaging pla o m om a dilu ion se ies o indocyanine g een (ICG)
dilu ed in dime hyl sul oxide (DMSO) a concen a ions anging om 25 nM o 800 nM in colo (le ), NIR2 fluo escence (middle) and a me ged image
o he colo wi h he fluo escence ( igh ); (B) plo showing he dilu ion se ies and a phan om backg ound imaged a a ious exposu e imes om 5 o
80 ms; (C) plo showing he da k noise and leakage wi h di e en binning, exposu e se ings and backg ound ma e ials.
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