YJPSU-59860; No of Pages 8

Journal of Pediatric Surgery xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Pediatric Surgery

journal homepage: www.elsevier.com/locate/jpedsurg

Review Article

Fluorescence imaging in pediatric surgery: State-of-the-art and

future perspectives
Irene Paraboschi a,b,c,⁎, Paolo De Coppi b,d, Danail Stoyanov a, John Anderson c,e, Stefano Giuliani a,d
a
Wellcome/EPSRC Centre for Interventional & Surgical Sciences, University College London, London, UK
b
Stem Cells & Regenerative Medicine Section, UCL Great Ormond Street Institute of Child Health, London, UK
c
Cancer Section, Developmental Biology and Cancer Programme, UCL Great Ormond Street Institute of Child Health, London, UK
d
Department of Specialist Neonatal and Pediatric Surgery, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK
e
Department of Oncology, Great Ormond Street Hospital for Children NHS Foundation Trust, London, England, UK

a r t i c l e i n f o a b s t r a c t

Article history: Background: The employment of fluorescence imaging has gained popularity in many fields of adult surgery
Received 29 April 2020 where it has demonstrated great potentials to improve both surgical and oncological outcomes while minimizing
Received in revised form 15 July 2020 anesthetic time and lowering health-care costs. However, the clinical application of fluorescence-guided surgery
Accepted 6 August 2020 (FGS) in pediatrics is just at the initial phase.
Available online xxxx
Material and methods: A systematic review of current clinical uses of FGS in pediatric surgery was performed
along with a discussion on its advantages, limitations and future developments.
Key words:
Fluorescence-guided surgery
Results: 21 studies were included: 9 retrospective and 1 prospective study, 8 case reports, 2 case series and a
Fluorescence imaging review article reporting authors' institutional experience. Great emphasis was given to surgical resection of
Near-infrared dyes hepatoblastoma and its metastasis (n = 6), real-time imaging of the biliary tree (n = 3) and urogenital sys-
Indocyanine green tem (n = 2). Other current uses concern the assessment of blood perfusion (intestine, n = 3; myocutaneous
Fluorescein sodium flap, n = 1; transplanted liver, n = 1) and lymphatic flow imaging (n = 4).
Conclusion: Despite a paucity of clinical studies evaluating its role in pediatric surgery, FGS has shown
promising results in helping guide tumor resection and improving the accuracy of anatomical delineation.
Type of study: Review article.
Level of confidence: Level IV.
© 2020 Elsevier Inc. All rights reserved.

Contents

1. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2. Current applications divided by anatomical and surgical areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.1. Imaging of the biliary tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.2. Vascular perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.3. Lymphatic flow imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.4. Tumor resection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2.5. Urogenital surgery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3.1. Designing fluorescent imaging probes with deeper tissue penetration and higher organ specificity . . . . . . . . . . . . . . . . . . . . . 0
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

⁎ Corresponding author at: Wellcome/EPSRC Centre for Interventional & Surgical Sciences, University College London, London, UK.
E-mail address: i.paraboschi@ucl.ac.uk (I. Paraboschi).

https://doi.org/10.1016/j.jpedsurg.2020.08.004
0022-3468/© 2020 Elsevier Inc. All rights reserved.

Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
2 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
In pediatric surgery there is an ever-increasing need for real-time in-
traoperative visualization of small anatomical structures and possible
congenital variants. Technological innovations, such as high-resolution
computed tomography (CT), magnetic resonance imaging (MRI) and
positron emission tomography (PET) have brought significant improve-
ments in the preoperative workup of surgical patients. Nevertheless,
their application in the operating room remains challenging as the
coregistration of preoperative images within the surgical field can be
difficult and may not always correspond to the definitive intraoperative
findings.
Nonionizing optical imaging techniques present a potentially ap-
pealing solution for all these problems [1]. In particular, the recent ad-
vent of fluorescence-guided surgery (FGS) has shown great potential
to improve surgical outcomes thanks to its ability to distinguish bile,
blood and lymphatic vessels and diseased from nondiseased tissues
[2–5], thus bridging the gap between preoperative imaging and intraop-
erative findings.
Although most of the current applications of FGS are mainly re-
ported for adult surgery, there has recently been an exponential in-
crease in the number of publications in pediatric surgery.
Therefore, we aimed to collect in a systematic fashion all the most
recent evidences on the use of fluorescence optical imaging in pediatric
surgery, producing simple guidelines on when there is a strong indica-
tion to use this novel technology in children while exploring its long-
term benefits for the treatment of several pediatric surgical conditions.
1. Methods
This review was performed according to the Preferred Reporting
Items for Systematic Reviews and Metanalysis Statement (http://
www.prisma-statement.org/). An extensive search was conducted in
the electronic database MEDLINE from inception through January
2020 using various combinations of keywords such as “Fluorescent-
guided” [All Fields] OR “Fluorescence-guided” [All Fields] AND “Surgery”
[All Fields]. Additional records were identified through hand-searching
the references reported in each selected articles. Two researchers (IP
and SG) carried out independent data extraction and quality assess-
ment. Any disagreement was resolved by consensus or by arbitration
of the other authors not involved in the initial procedure.
The inclusion criteria for the systematic review were original studies,
written in English, that reported the clinical use of FGS for the surgical
treatment of children (0–18 years old) affected by thoracic, abdominal
or urogenital diseases or tumors. Preclinical studies, review papers not
reporting own clinical experience and studies employing fluorescent
dyes for diagnostic purposes only were excluded, as well as articles de-
scribing neurosurgical, vascular, ophthalmological, ENT, maxillofacial or
aesthetic surgical procedures.
An electronic database (Microsoft Excel 2007, Redmond, WA, USA)
was prepared to collect the following information: authors, article
title, journal, year of publication, study period, study design, sample
size, age at surgery, underlying diseases, type of surgical procedure,
complications, type of fluorophore selected and imaging system
adopted.
Owing to the heterogeneity and the small number of studies a meta-
analysis of the available data was deemed unfeasible.
2. Results
2.1. Overview
As shown in the Prisma flow diagram (Fig. 1), 828/849 studies were
excluded. 499 were excluded on a title basis, and 220 were excluded on
an abstract basis. 130 studies were evaluated on a full-text basis. Out
of them, 109 had to be excluded because they were focused on adult
population (n = 83), preclinical studies (n = 14) or review articles
(n = 12).
Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
Therefore, 21 studies were included in the systematic review. 9/21
(42.9%) were retrospective studies, 8/21 (38.1%) were case reports
and 2/21 (9.5%) were case series. There was only a single prospective
study investigating the role of fluorescein sodium in suspected necrotiz-
ing enterocolitis (NEC) cases [6]. Finally, Yamada et al. [7] published a
review article on ICG imaging for hepatoblastoma (HB) patients, de-
scribing at the same time their extensive institutional experience in
detail.
Table 1 summarizes the current clinical applications of FGS in gen-
eral pediatric surgery: 3/21 (14.3%) articles focused on real-time imag-
ing of the biliary tree, 5/21 (23.8%) on vascular perfusion, 4/21 (19.0%)
on lymphatic flow, 6/21 (28.6%) on tumor resection and 2/21 (9.5%)
on urogenital surgery. A single article (4.8%) described a unicentric ex-
perience of laparoscopic FGS in children, including one cholecystec-
tomy, two radical nephrectomies and one varicocelectomy.
Indocyanine green (ICG) was the dye employed in 20 (95.2%) out of
21 articles and fluorescein sodium in the remaining one.
As shown in Table 1, the fluorescent dyes were administrated fol-
lowing different protocols depending on the authors' choice and clinical
indications. For laparoscopic cholecystectomy, ICG was intravenously
injected from 15 min [8] to 18 h [9] prior to surgery, according to differ-
ent operative strategies. For Kasai hepatoportoenterostomy (HPE) the
same timing of ICG injection was adopted, however with two different
concentrations (0.5 mg/kg [10] and 0.1 mg/kg [11]). Conversely, for
HB resection, the same ICG dose was employed (0.5 mg/kg) but with
different timing (from 24 h [12,13] to 72 h [7,14] prior to surgery).
With regards to drug safety profile, no side effects were reported
with the administration of ICG and fluorescein sodium. In particular,
no allergic reactions and no alterations in heart rate, blood pressure or
respiratory status occurred.
Focusing on optical imaging devices, the fluorescence signal was de-
tected by cameras marketed by Hamamatsu Photonics in 6/21 (28.6%)
studies, Karl Storz in 5/21 (23.8%), Stryker in 2/21 (9.5%), Mizuho Med-
ical Co and Novadaq Technologies in 1/21 (4.8%) study each. Chen-
Yoshioka et al. [12] developed a new optical imaging system (the Med-
ical Imaging Projection System, MIPS) in association with Panasonic
AVC Networks Company. 5/21 (23.8%) authors didn't disclose the imag-
ing system employed.
2.2. Current applications divided by anatomical and surgical areas
2.2.1. Imaging of the biliary tree
In total 4 (19.1%) articles employed ICG for the intraoperative assess-
ment of the biliary tree: 2 papers for guiding pediatric cholecystectomy
[8,9] and 2 studies for Kasai HPE (n = 2) [10,11].
In particular, in 2019, Esposito et al. [9] compared the results of 15
ICG vs 200 traditional laparoscopic cholecystectomies performed in
their center in the last 25 years. As expected, the enhanced mapping
of the biliary tree helped to shorten the average operative time (52 vs
69 min) and reduce the postoperative complication rate (0 vs 1.9%) in
the group of patients undergoing ICG cholecystectomy (Fig. 2). Simi-
larly, ICG delineated well the biliary anatomy during a laparoscopic cho-
lecystectomy performed in a 13-year-old girl with a long history of
recurrent cholelithiasis, providing the utility of this technology in com-
plex cases as reported by Fernandez-Bautista et al. [8].
ICG has also proved to be particularly helpful for the delicate dissec-
tions of the proximal biliary tree, such as in case of Kasai HPE for infants
with biliary atresia (BA). In this regard, ICG-FGS was firstly described by
Hirayama et al. [11] as an essential tool for real-time detection of bile ex-
udation from the porta hepatis after dissection of the fibrous tissue.
These authors mostly provided qualitative results suggesting that the
intraoperative ICG cholangiogram led to better identification of the bil-
iary structures with clear visualization of the fibrous cone. This helped
the appropriate level and extent of dissection, therefore resulting in suc-
cessful surgeries with good long-term clinical outcomes.
 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx 3

PRISMA 2009 Flow Diagram

Idenficaon

Records idenfied through Pubmed Addional records idenfied

database searching through other sources
(n = 830) (n = 19)

Records aer duplicates removed

(n = 849)
Screening

Records screened Records excluded

(n = 849) (n =719)

Full-text arcles excluded,

with reasons
Eligibility

Full-text arcles assessed

for eligibility (n =109)
(n = 130) - Arcles focusing
on adult
populaon, n=83
- Preclinical studies,
n=14
- Review arcles,
Studies included in n=12
qualitave synthesis
Included

(n = 21)

Fig. 1. PRISMA 2009 flow diagram.

More quantitative analyses were later reported by Yanagi et al. [10],
who compared the jaundice outcomes of 10 patients undergoing ICG-
FCG with 35 historical patients who underwent traditional HPE or
hepaticojejunostomy. Despite the small number of patients, all children
in the first group postoperatively normalized their bilirubin levels,
whereas 12 (34.3%) children in the second group did not (p-value
<0.05). They claimed that this result was achieved by the enhanced vi-
sualization of the hilar micro bile ducts during the surgical dissection.
2.2.2. Vascular perfusion
In total 5 (23.8%) articles described fluorescent angiography for
assessing vascular perfusion during intestinal surgery (n = 3)
[6,15,16], liver resection (n = 1) [17] or plastic reconstruction (n = 1)
[18].
Numanoglu et al. [6] were the first to report the employment of fluo-
rescein sodium dye during diagnostic laparoscopy of 8 premature in-
fants with suspected NEC. The fluorescein-aided assessment allowed
the detection of 3 patients with ischemic bowel segments which were
not visible with the naked eye. The dye-enhanced bowel was dissimilar
from the ischemic one, which appeared dark under UV light with a yel-
low filter.
More recently, other authors have reported their experience by
using ICG angiography for the intraoperative assessment of intestinal
perfusion in children.
In particular, complex reconstructive intestinal surgeries have
benefited from the employment of fluorescent intraoperative perfusion
assessment techniques, as reported by Rentea et al. [15] who investi-
gated the role of ICG fluorescence angiography to assess rectal and
neovaginal pull-throughs in cloacal reconstructions (n = 9), complex
anorectal malformation (ARM) (n = 1) and Hirschsprung disease
(HD) (n = 3) repairs. They stated that fluorescent imaging significantly
impacted the intraoperative decision-making process in a third of their
patients, influencing the decision to redo an intestinal anastomosis
(n = 1), choose a more proximal resection margin (n = 2) and resect
a portion of bowel (n = 1).
Moreover, Iinuma et al. [16] described the case of a 15-year-old boy
undergoing a massive necrotic intestinal resection owing to a small
bowel volvulus. Although ICG angiography had shown an abnormal
vascular flow pattern in the distal part of the residual jejunum, im-
provements in the clinical findings of the intestine supported the sur-
geons' decision on performing a primary anastomosis. However, a
partial stricture developed on the intestinal segment affected by the

Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
4 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx

Table 1
Current literature focusing on fluorescence-guided surgery (FGS) in general pediatric surgery.

Author, year Type of study Disease treated (no. of Type of surgery performed (no. of Study Patients' age Dye (administration Imaging
patients affected) patients per type of procedure) period at surgery route and dosage) system,
company

Anatomic imaging of the biliary tree

Esposito et al., 2019 Retrospective Cholelithiasis (n = 15) Laparoscopic cholecystectomy 2016–2018 nd ICG (iv, 0.4 mg/kg, 18 h Image1 S™,
[9] study (n = 15) before surgery) Karl Storz
Yanagi et al., 2019 Retrospective Biliary atresia (n = 10) Kasai 2017–2018 Median: 69.5 ICG (iv, 0.5 mg/kg, 24 h Vitom®, Karl
[10] study hepatoportoenterostomy(n = 9); d before surgery) Storz
hepaticojejunostomy (n = 1) (48–122 d)
Hirayama et al., Retrospective Biliary atresia (n = 5) Kasai hepatoportoenterostomy 2012–2014 Median: 42 d ICG (iv, 0.1 mg/kg, 24 h Photodynamic
2015 [11] study (n = 5) (31–75 d) before surgery) Eye,
Hamamatsu
Photonics

Vascular perfusion
Rentea et al., 2019 Retrospective Cloaca (n = 9), rectal Posterior sagittal 2014–2018 Mean: 1.9 yrs ICG (iv, 0.1–0.3 mg/kg, SPY Elite
[15] study atresia (n = 1), anorectovaginouretroplasty (0.5–7.8 yrs) at time of surgery) Imaging
Hirschsprung disease (PSAVUP) (n = 8), redo PSARVUP System,
(n = 3) (n = 1), colonic pull-through Stryker
(n = 1), redo pull-through
(n = 3)
Kisaoglu et al., Case report Focal hepatic necrosis Liver resection nd 4 yrs ICG (iv, 0.05 mg/kg, at nd
2019 [17] after liver transplantation time of surgery)
owing to maple syrup
urine disease (n = 1)
Fried et al., 2019 Case report Teratoma (n = 1) Free latissimus dorsi nd 6 mo ICG (nd) nd
[18] myocutaneous flap
Iinuma et al., 2013 Case report Small intestinal volvulus Primary intestinal anastomosis 2013 15 yrs ICG (iv, 25 mg, at time Photodynamic
[16] (n = 1) (n = 1) of surgery) Eye,
Hamamatsu
Photonics
Numanoglu et al., Prospective Suspected necrotizing Diagnostic laparoscopy followed nd 24.5 d Fluorescein (iv, Karl Storz
2011 [6] study enterocolitis (n = 8) by bowel resection and stoma (10–38 d) 14 mg/kg, at time of
formation (n = 8) surgery)

Lymphatic flow imaging

Shirotsuki et al., Retrospective Esophageal Diagnostic thoracoscopy (n = 8), 2014–2017 Median: 2.0 d ICG (sc, 0.025 mg, 1 h Image1 S™,
2018 [20] study atresia/tracheoesophageal thoracoscopic ligation of the (1.0–10 d) prior to surgery) Karl Storz
fistula (n = 10) injured thoracic duct (n = 3) Median: 18 d
(13–25 d)
Shirota et al., 2017 Case report Lymphatic malformation Surgical resection 2013 15 yrs ICG (sc and id, Photodynamic
[19] of the abdominal wall 0.125 mg, 20 h prior to Eye (PDE),
(n = 1) surgery) Hamamatsu
Photonics
Tan et al., 2014 [21] Case report Iatrogenic chylothorax Diagnostic imaging of the 2014 5 wks ICG (id, 1st injection 25 nd
(n = 1) lymphatic flow followed by μg, 2nd injection 12.5
bilateral pleurodesis μg 19 min later, 3rd
injection 12.5 μg
26 min later)
Chang et al., 2014 Case report Iatrogenic chylothorax Intraoperative lymphography and nd 3 mo ICG (sc, 2 ml, at time of nd
[22] (n = 1) lymphatic fistula closure surgery)

Tumor resection
Souzaki et al., 2019 Retrospective Primary HB (n = 3), HB Extended right hepatectomy 2017–2018 Mean: 30.9 ICG (iv, 0.5 mg/kg, D-LIGHT P,
[23] study lung metastases (n = 1), (n = 3), liver transplantation mo (12–36 60–138 min prior to Karl Storz
primary and HB lung (n = 1), lung partial resection mo) liver surgery, 18–27 h
metastases (n = 1) (n = 5), upper lobectomy prior to lung
(n = 1) metastatectomy)
Takahashi et al., Case report HB peritoneal Surgical excision (n = 1) nd 14 yrs ICG (iv, 0.5 mg/kg, 72 h Photodynamic
2019 [14] dissemination (n = 1) prior to surgery) Eye (PDE),
Hamamatsu
Photonics
Yamada et al., 2019 Review study Primary HB (n = 12), HB Liver resection (n = 13), lung 2014–2019 Mean: 5.0 yrs ICG (iv, 0.5 mg/kg, 72 h Photodynamic
[7] lung metastases (n = 7), metastasectomies (n = 15), other (0.5–14 yrs) prior to surgery Eye (PDE),
mediastinal metastasis metastasectomies (n = 5) Hamamatsu
(n = 1), peritoneal Photonics
metastasis (n = 1),
pancreatic metastasis
(n = 1), bone metastasis
(n = 1)
Chen-Yoshikawa Case report HB lung metastasis Lung metastatectomy (n = 1) nd 3 yrs ICG (0.5 mg/kg, 24 h Medical
et al., 2017 [12] (n = 1) prior to surgery Imaging
Projection
System,
Panasonic AVC
Networks
Company

Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx 5

Table 1 (continued)

Author, year Type of study Disease treated (no. of Type of surgery performed (no. of Study Patients' age Dye (administration Imaging
patients affected) patients per type of procedure) period at surgery route and dosage) system,
company

Kitagawa et al., Retrospective HB lung metastases Lung metastatectomies (n = 37) 2012–2014 Mean: 3.5 yrs ICG (iv, 0.5 mg/kg, 24 h Photodynamic
2015 [13] study (n = 10) (1–11 yrs) prior to surgery) Eye,
Hamamatsu
Photonics
Yamamichi et al., Case series Primary HB (n = 1), Right hepatectomy (n = 1), nd Mean: 3 yrs ICG (iv, 0.5 mg/kg, HyperEye
2015 [24] recurrent HB (n = 1), HB residual tumor and diaphragm (1–6 yrs) 72–96 h prior to Medical
lung metastasis (n = 1) resection (n = 1), lung surgery) System,
metastasectomy (n = 1) Mizuho
Medical Co

Urogenital surgery
Esposito et al., 2019 Retrospective Varicocele (n = 25) Laparoscopic Palomo 2017–2018 Mean: ICG (intratesticular nd
[25] study varicocelectomy (n = 25) 13.7 yrs 0.1 mg, at time of
(12–16 yrs) surgery)
Herz et al., 2016 Retrospective Duplex kidney (n = 6) Robot-assisted laparoscopic 2014–2016 Mean: 5.6 yrs ICG (iv, 1.25–2.5 mg, Firefly, da
[26] study heminephrectomy (n = 6) (0.8–13.2 yrs) 30–60 s prior to Vinci, Novadaq
surgery) Technologies
Inc.

Articles reporting multiple surgical diseases treated with FGS

Fernàndez-Bautista Case series Cholelithiasis (n = 1); Laparoscopic cholecystectomy nd Mean: 8.6 yrs ICG (iv, nd, 15 min 1488 HD
et al., 2019 [8] varicocele (n = 1); renal (n = 1); laparoscopic Palomo (3–13 yrs) before laparoscopic 3-Chip camera
failure (n = 2) varicocelectomy (n = 1); cholecystectomy; iv, nd, system,
laparoscopic nephrectomy at the time of Stryker
(n = 2) laparoscopic Palomo
varicocelectomy; iv,
0.2 mg/kg, at time of
laparoscopic
nephrectomy)

ICG: indocyanine green; HB: hepatoblastoma; iv: intravenously; sc: subcutaneously; id: intradermal; yrs: years; mo: months; d: days; h: hours; min: minutes; s: seconds; nd: not defined.

atypical angiographic findings, which required a surgical revision two
weeks later.
Moving beyond, in 2019, Kisaoglu et al. [17] described the use of the
ICG fluorescence imaging for identifying and managing insufficient graft
perfusion in a 4-year-old boy who underwent liver transplantation.
In addition, Fried et al. [18] described the successful case of a 6-
month-old child who required a free latissimus dorsi flap reconstruction
after the surgical resection of an immature teratoma of the temporal
area. The superior thyroid artery and the retromandibular vein were se-
lected as the new blood supply for the graft. Laser-assisted ICG assess-
ment after the vascular anastomosis added invaluable information
about the good perfusion of the graft.
2.2.3. Lymphatic flow imaging
In total 4 (19.1%) articles described the use of ICG for real-time imag-
ing of the lymphatic flow: a single paper for guiding the radical excision
of a congenital lymphatic malformation [19] and 3 studies for
preventing or identifying iatrogenic thoracic duct injuries [20–22].
In detail, Shirota et al. [19] described the successful resection of a
lymphatic malformation of the abdominal wall in a 15-year-old boy
guided by the intradermal and subcutaneous administration of
0.125 mg of ICG injected 20 h prior to surgery.
Shirotsuki et al. [20] collected a series of 11 procedures in which
0.025 mg of ICG was injected 1 h prior to surgery in 10 infants with
esophageal atresia/tracheoesophageal fistula (EA/TEF). In 8
thoracoscopic procedures ICG was preoperatively administered to de-
tect the thoracic duct and to prevent iatrogenic lesions at the time of
the primary repair, while in the 3 remaining cases to treat postoperative
chylothorax.
Fluorescence lymphatic imaging also allowed the successful visuali-
zation of abnormal lymphatic drainage in two infants who developed
postoperative chylous leakages after congenital heart surgery.
In particular, Tan et al. [21] described the employment of ICG lym-
phography to guide the therapeutic strategy in a 5-week-old infant
with hypoplastic left heart syndrome who developed a postoperative
left chyothorax. As result of the accumulation in the left pleural cavity
of 12.5 μg of ICG administered in the ipsilateral hand, a left pleurodesis
was successfully performed in coincident to the decrease in left chest
tube drainage.
Similarly, the chylous leakage point responsible for a massive post-
operative chylothorax, refractory to both conservative (fasting, total
parenteral nutrition) and invasive (two thoracotomic thoracic duct liga-
tions) treatments, was clearly identified 20 min after the inguinal injec-
tion of 2 mL of ICG in a 3-month infant with an endocardial cushion
defect [22].
2.2.4. Tumor resection
In total 6 (28.6%) articles explored the application of ICG navigation
imaging for pediatric HB resection [7,12–14,23,24].
Yamamichi et al. [24] ran the first pilot study evaluating the benefits
of ICG navigation surgery for identifying small viable HB tumors. A pri-
mary HB of the right lobe, a recurrent HB located between the right di-
aphragm and the cut surface of the previous surgery and bilateral
multiple lung metastases were clearly visualized and safely excised
three or four days after the intravenous injection of ICG (0.5 mg/kg).
The same year, Kitagawa et al. [13] published the resection of 250
fluorescence-positive pulmonary lesions from 10 patients affected by
metastatic HBs (mean age: 4 years; range: 4 months–11 years and
4 months). The visual fluorescence contrast of the metastatic lesions af-
fecting the peripheral areas of the lung along with the intraoperative
collapse of the lung allowed by the one-lung ventilation system led to
precise metastatic tumor resections 24 h after the intravenous injection
of 0.5 mg/kg of ICG.
Moreover, four years later, Takahashi et al. [14] described the case of
a 14-year-old-boy who successfully underwent an open
metastasectomy of peritoneally disseminated HBs with ICG navigation
followed by a second living donor liver transplantation (LDLTx). The
Photodynamic Eye system (Hamamatsu Photonics, Hamamatsu,
Japan) was used to visualize the tumor lesions taking up ICG, intrave-
nously injected 72 h prior to surgery at a dosage of 0.5 mg/kg.

Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
6 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
Fig. 2. ICG fluorescence enabled an easy identification of the cystic duct despite the presence of abundant fatty tissue or fibrotic adherences.
Charge-coupled device (CCD) and other types of cameras have been
safely employed for ICG detection during HB surgery in children. For in-
stance, Chen-Yoshioka et al. [12] developed the Medical Imaging Projec-
tion System (MIPS, Panasonic AVC Networks Company, Osaka, Japan),
which was able to detect fluorescence signals of lung HB metastases
24 h after ICG injection even with the operating lights turned on and
to project the image in visible light back onto the organ being operated
on.
More recently, Souzaki et al. [23] published the largest series of 5 pa-
tients undergoing 10 ICG surgeries (n = 4 open liver resections, n = 5
open lung metastasectomies, n = 1 thoracoscopic-assisted lung
metastasectomy) by using a 10-mm endoscopic ICG NIRF imaging sys-
tem (D-LIGHT P, Karl Storz, Germany). The telescopic detector had the
advantages of keeping the scope tip closer to the tumor and of perfectly
darkening the surgical field. All these elements led to an increased sur-
gical accuracy with a better appreciation of the tumor margins and of its
spread to the surrounding structures.
Finally, Yamada et al. [7] published their unicentric institutional ex-
perience, which includes 13 laparotomies for 12 children with primary
tumors of the liver, 15 thoracotomies for 7 patients with pulmonary me-
tastases and 5 surgeries for 4 patients with lymph-node metastasis
(n = 1), peritoneal metastasis (n = 2), pancreatic metastasis (n = 1)
and bone metastasis (n = 1). They also analyzed current literature on
this topic, proposing protocols for fluorophore administration, tumor
imaging and surgical excision.
2.2.5. Urogenital surgery
In total 3 (14.3%) articles reported the adoption of fluorescent optical
imaging in pediatric urology [8,25,26].
Initially described for segmental arterial mapping during pediatric
robot-assisted laparoscopic heminephrectomy (RALHN) [26], ICG injec-
tion was also employed to enhance the visualization of blood [8] and
lymphatic [25] vessels during laparoscopic Palomo varicocelectomy.
In particular, ICG-FGS proved to be a valuable tool in reducing the in-
cidence of innocent moiety injury in 6 pediatric RALHNs in the series re-
ported by Herz et al. [26]. Real-time delineation of the selective arterial
anatomy of both moieties was performed safely without any toxicity or
vascular complications. ICG-FGS helped in alerting surgeons about un-
expected renal vascular anatomy and possible iatrogenic injuries to
the remaining moiety, thus adding invaluable information during
surgery.
Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
In addition, ICG optical imaging was also safely employed to accu-
rately study the renal vascular anatomy in two patients undergoing ret-
roperitoneal laparoscopic nephrectomies in the series collected by
Fernandez-Bautista et al. [8].
The same authors [8] reported the use of an intravenous injection of
ICG to perform an angiography-assisted laparoscopic varicocelectomy
in a 13-year-old boy with asymmetric testes and testicular pain.
Conversely, Esposito et al. [25] described ICG fluorescence lymphog-
raphy as a new technique to perform lymphatic sparing laparoscopic
varicocelectomy: none of the 25 boys reported in their series developed
a postoperative hydrocele.
3. Discussion
Fluorescent optical imaging has proved to be a safe tool to guide sev-
eral surgical procedures in children. The great spatial resolution of even
fine anatomical structures, the high contrast and sensitivity, the absence
of ionizing radiations and the low cost are the main advantage of this
novel technique, which employs fluorescent dyes and optical imaging
devices for the real-time assessment of the surgical field.
With regards to fluorescent probes, both IGC and fluorescein sodium
have been used in children, with no reported side-effects.
ICG is an anionic amphiphilic tricarbocyanine dye approved by the
Food and Drug Administration (FDA) for more than 60 years [8]. By
binding albumin, IGC is normally confined into the vascular stream
and entirely excreted into the biliary tract within a few hours from in-
jection. Water-solubility and fast biliary secretion in normal liver
make the visualization of vascular perfusion and biliary flow among
the most common clinical applications of ICG in children. Conversely,
its delayed excretion in neoplastic hepatocytes makes ICG a promising
tool for surgical margin definition during primary and metastatic HB
resections.
Fluorescein sodium is a safe and inexpensive water-soluble dye, cur-
rently used as a diagnostic tool for retinopathy in preterm infants, and
its utility in the assessment of ischemic bowels and intracranial tumors
has been known since 1942 [27] and 1947 [28], respectively.
Several clinical applications of these two fluorophores have been de-
scribed in pediatric surgery, ranging from vascular assessment to biliary
and lymphatic imaging, from oncology to urogenital procedures.
Based on the data presented, we would recommend the used of in-
traoperative fluorescence for complicated ‘adult-type’ laparoscopic cho-
lecystectomies, for the assessment of bowel perfusion during complex
 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
colorectal procedures, for microvascular reconstructions in plastic sur-
gery and for selective arterial mapping in pediatric urology.
Laparoscopic cholecystectomy is often a smoother operation in chil-
dren compared to adult cases where dilatation, inflammation and scar
tissue of the biliary tree can make the dissection challenging for the sur-
geon. However, the presence of anatomical variants of the cystic duct
(1% in the series of laparoscopic cholecystectomies reported by Carbajo
et al. [29]) and ‘adult-like’ gallbladders can make this procedure a real
nightmare for pediatric surgeons, especially for those with a low-
volume of laparoscopic cases. In this setting, we believe the use of ICG
should become the gold standard as it can support the view and reduce
the risk of injury of the biliary tree [8,9].
Fluorescent optical imaging has also proved to be a safe and effective
method for real-time assessment of vascular supply during complex ab-
dominal procedures. In a systematic review of the literature, ICG-FGS
has significantly reduced the risk of anastomotic leakages in comparison
with conventional imaging during colorectal surgeries in adults [30],
aiding in decision-making as to where to resect the intestinal segment.
The assessment of an adequate blood supply is also pivotal in pediatric
colorectal and neovaginal pull-throughs. Maintaining a good vascular
pedicle without tension or kink at the site of the anastomosis is crucial
for the surgical repairs of cloaca, high ARMs and long-segment HDs,
which all depend on the vascular supply coming from the mid or right
colic artery. In this respect, although for a definitive clinical validation
in children some points still required further investigations and quanti-
tative data analysis (eg. vascular signal measurements, perfusion index
and pixel intensity curves), very promising results have been achieved
so far [15].
ICG angiography has also been described as meaningful tool for
assessing tissue viability during microsurgical plastic reconstructions
in infants. By providing an efficient magnification method for real-
time visualization of small vessels of the recipient site, ICG vascular as-
sessment has overcome one of the main critical issues for free flap re-
constructions in pediatric settings, reducing at the same time age
limits and postoperative complications.
In the field of pediatric urology, unexpected vascular anatomy, ex-
tensive traction injury during a complex dissection, involuntary disper-
sion of cautery energy may all complicate minimally invasive
procedures. In this respect, a safer dissection at the level of the renal
hilum and spermatic cord can be easily performed by the intraoperative
injection of ICG, which allows renal and spermatic vessels to be identi-
fied with certainty. As reported by Herz et al. [26] and Fernández-
Bautista et al. [8], ICG selective arterial mapping prevented the risk of
damaging the ipsilateral innocent renal moiety during laparoscopic
heminephrectomy (estimated at 4.9% [31]) and the occurrence of intra-
operative complications during laparoscopic retroperitoneal nephrec-
tomy (estimated at around 4.3% [32]). Likewise, no varicocele
recurrences (estimated at 7%–35% [33]) and no postoperative hydro-
celes (estimated at 10%–30% [25]) occurred in the cohort of patients un-
dergoing ICG-assisted laparoscopic Palomo varicocelectomy, as
described by Esposito et al. [25] and Fernández-Bautista et al. [8].
However, it is especially in the oncological field that FGS has been
emerging as a cutting-edge innovation in which very accurate margin
definitions can lead to more radical tumor resections with improved
surgical outcomes [2,7,12–14,23,24]. In fact, tumor-specific fluores-
cence probes can be employed to selectively light up active tumor
cells, decreasing the rate of incomplete tumor resections and helping
spare uninvolved healthy tissue or necrotic/scarred tumor cells
postchemotherapy.
Although ICG navigation imaging for pediatric HB is currently at its
initial stages, early results seemed to be very promising [7,12,14,23].
In the near future, the accumulation of more HB cases will lead to
more uniform procedural protocols for establishing the optimal timing
and dosage for ICG injection and for a more accurate patients' selection
with regards to tumor depth from liver surface. In fact, despite the var-
ious benefits of ICG-based fluorescent imaging in hepatobiliary surgery,
Please cite this article as: I. Paraboschi, P. De Coppi, D. Stoyanov, et al., Fluorescence imaging in pediatric surgery: State-of-the-art and future
perspectives, Journal of Pediatric Surgery, https://doi.org/10.1016/j.jpedsurg.2020.08.004
7
one of the current limitations for its clinical use is related to the limited
tissue penetration of near-infrared light. Further developments in opti-
cal imaging may soon overcome this limit, providing fluorescent dyes
and detection technologies with a greater tissue penetration. Moreover,
the incorporation of FGS to thoracoscopic devices will favorably supple-
ment the tactile-limited field of minimally invasive surgery, improving
the cosmetic and functional outcomes in infants undergoing HB metas-
tasis lung resections.
Despite very promising reports [10,11], further prospective studies
with a larger cohort of patients involved are warranted for validating
ICG-FGS as the gold standard navigation tool for Kasai HPE in infants
with BA. In particular, the ideal timing of ICG injection has yet to be
established to achieve the greatest fluorescent contrast between biliary
structures and liver parenchyma with the aim to provide meaningful
objective information about the level of transection at fibrous cone
that can integrate subjective surgeon's impression.
Likewise, even if early reports seem to be very encouraging in prov-
ing the utility of ICG for navigation of lymphatic malformations [19] and
iatrogenic thoracic duct injuries [20–22], the small patient sample size
and the retrospective nature of the studies (mainly case reports) repre-
sent the main limits for large-scale adoption of ICG-lymphography.
Further studies involving more patients and continued experience
with fluorescent imaging are also needed for definitive validation of
fluorescein laparoscopy as a meaningful tool for identifying necrotic
segments in infants with suspected NEC [6].
3.1. Designing fluorescent imaging probes with deeper tissue penetration
and higher organ specificity
Although the past decade has no doubt witnessed significant ad-
vances in the clinical application and technical development of fluores-
cent optical imaging, there is still room for further developments.
To date, biomedical fluorescence imaging has mainly relied on near
infrared-I (NIR-I, wavelength: 700–900 nm) dyes, which has been fa-
vored over visible light (wavelength: 380–800 nm) owing to less tissue
autofluorescence and absorbance.
In this regards, the high safety index, the good pharmacokinetic pro-
file and the low cost of ICG have made it one the most useful NIR-I dyes
for proper visualization of the blood, biliary and lymphatic flow in
children.
However, the low tissue penetration (up to 10 mm) and limited tis-
sue contrast of NIR-I dyes have limited their surgical applications, also in
children. In this respect, more recent studies are investigating NIR-II
(wavelength: 1000–2000 nm) fluorophores as promising tools for
achieving higher contrast, greater sensitivity and improved penetration
depths [34].
Although no NIR-II fluorophores have been approved for clinical use
in humans yet, the discovery that some NIR-I dyes (such as ICG) display
bright emission tails over 1000 nm offers exciting opportunities for en-
hanced surgical imaging, especially in the field of surgical oncology.
Repurposing clinically approved NIR-I fluorescent probes as NIR-II
agents shows important clinical implications, as reported by Hu et al.
[35] who firstly developed an integrated visible and NIR-I/NIR-II multi-
spectral imaging instrument for ICG-guided surgical resection of hepa-
tocellular carcinomas (HCCs).
To maximize the signal from tumor cells and to minimize back-
ground noise not only NIR-II fluorophores but also tumor-targeted
fluorescent probes are currently under investigation [2]. In particu-
lar, promising results have been achieved in clinical trials of adult
oncology exploring the efficacy of fluorescently labeled monoclonal
antibodies to detect viable tumor cells and to better define surgical
margins. Both anti-EGR and anti-VEGF monoclonal antibodies have
been successfully conjugated with IRDye800CW for targeting head
and neck, colon and breast cancers and pancreatic adenocarcinomas
in adult patients [2,3].
8 I. Paraboschi et al. / Journal of Pediatric Surgery xxx (xxxx) xxx
Similar strategies can be also adopted for pediatric cancers for which
tumor-specific monoclonal antibodies have been clinically approved.
In this regards, one of the main limits of fluorescently labeled mono-
clonal antibodies, which is the slow quenching process, will be soon
overcome by new sprayable targeted probes. They will be applied into
the region of interest by a simple spray, making their clinical use very
convenient [2].
Finally, complementary analytical tools, such as artificial intelligence
(AI), will be integrated into an FGS system to improve the decision-
making capability of fluorescence optical imaging [36].
4. Conclusion
In recent years, FGS has brought forth new lights in pediatric sur-
gery. In particular, ICG optical imaging has proved to be a safe and effi-
cient tool for improving the accuracy of biliary flow delineation during
pediatric laparoscopic cholecystectomies and bowel perfusion during
complex colorectal procedures. Microvascular reconstructions in plastic
surgery and selective arterial mapping in urogenital surgeries are other
interesting applications of FGS in children. Further studies involving
larger cohorts of patients and continued experience with fluorophores
and optical imaging system will also help FGS develop as a valuable nav-
igation tool for the definition of HB margins and clear visualization of
the fibrous cone during Kasai HPE. Lymphatic flow evaluation and
bowel perfusion assessment in neonates with suspected NEC are other
promising applications of fluorescent optical imaging in children.
Acknowledgment
This work was supported by the Medical Research Council (grant
number MR/T005491/1) and by the Wellcome/EPSRC Centre for Inter-
ventional and Surgical Sciences at University College London.
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