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L. Baldari et al.
near-infrared photography by the Kodak Research Laboratories
in 1955 and was approved for clinical use already in 1956.
However, it took over 10 years before ICG was used for angiography. It has been used for retinal angiography since the
early 1970s [36].
Indocyanine green is a negatively charged ion that belongs
to the large family of cyanine dyes, having a molecular weight
of 751.4Da. Dry ICG is stable at room temperature. In aqueous solutions, ICG molecules tend to aggregate, which inuences their optical properties. The aggregation depends on
concentration and time. The spectral stabilization is fastest
when ICG is dissolved in distilled water, and thus some authors
do not recommend adding isotonic saline and/or albumin to the
injectate, when fast spectral stability is essential, for example,
when using ICG for quantitative purposes. IGC is hydrophobic
and, thus, it frequently bounds to proteins in plasma (especially albumin), which connes ICG to the intravascular space
and makes it especially suited for angiographic applications
[26, 27]. ICG based angiography and lymphography have been
used in a variety of clinical indications, such as perfusion based
imaging of the liver and blood vessels of the eye and assessment of lymphatic vessel drainage. The important property of
fast binding to plasma proteins, especially lipoproteins, makes
repeated intraoperative applications of ICG possible. The binding to plasma proteins does not seem to alter protein structures,
which is one sign of nontoxicity. Binding to blood proteins
also shifts slowly, taking several minutes, the absorption peak
at 780nm toward longer wavelengths, to 805nm. The absorption peak maximum was observed at 810nm in the epidermal
cell cultures and at 805–810 nm in the human skin in vivo
(Figs.1.4 and 1.5).
ICG does not have any known metabolites, and it is quickly
extracted by the liver into bile juice. The transport is done by a
protein called glutathione S-transferase without modication.
The protein spectra of different liver diseases also affect ICG
protein binding in blood. It has a quick clearance rate of
18–24% per minute by the liver, which is the result of both the
0
Wavelength (nm)
Absorption / Emission (Normalized)
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Fig. 1.4 Chemical composition of indocyanine green
1.0
0.8
0.6
0.4
17
0.2
0
600 700 800 900 100
Fig. 1.5 Absorbance and uorescence-emission spectra of indocyanine
green. Continuous and dotted lines show absorbance and emission spectra
respectively
compound’s connement to the intravascular space and that
the decomposition products of ICG are not metabolites. The
dye is cleared from the system exponentially in the rst
10–20 min after application, with a half-life of generally
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L. Baldari et al.
3–4min depending on the vascularization of the organ of interest [26, 27, 36].
The typical dye concentrations used for invivo retinal angiography are in the range of 20–25mg/mL of ICG applied by
injection into a peripheral arm vein. For studies of hepatic
function, an intravenous injection dose is calculated on the
basis of 0.5mg/kg of body weight. In cardiac output and blood
volume monitoring, the total dose of dye injected should be
kept below 2mg/kg. Peculiar feature is the low toxicity (LD50
after single IV dose of 50–80mg/kg for animals). No signicant toxic effects have been observed in humans with the high
dose of 5mg/kg of body weight. ICG for injection contains
sodium iodide and should be used with caution in patients who
have a history of allergy to iodides because of the risk of anaphylaxis. ICG was not found to be mutagenic in the tests performed. No studies for reproduction, teratogenicity, or
carcinogenic properties in animals are available, but decades
of experience in humans have not revealed any incidence of
these properties.
To date, ICG is the first and only clinically approved
fluorophore that displays NIR fluorescence. Because ICG
has no functional groups for conjugation to targeting moieties for molecular imaging application, it is a nonspecific
contrast agent. The commercially available instrumentation used for ICG detection is adjusted for the characteristics that ICG displays in plasma (peak excitation
wavelength of 807nm and peak emission wavelength of
822nm) [36, 37].
• 5-aminolevulinic acid. 5-ALA is an amino acid that acts as a
uorophore processed by high metabolic active cells. Indeed, it
is a precursor of protoporphyrin IX that is involved in the pathway of heme synthesis. After 5-ALA administration, protoporphyrin IX accumulates in malignant glioma tissue, which
became uorescent, due to an abnormality in porphyrin metabolism.
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Fig. 1.6 Chemical composition of 5-aminolevulinic acid
It exhibits a uorescence absorption peak of 405nm, and an
emission peak in the red visible light range (635nm), permitting uorescence imaging on the surface of tissues and in a
depth range of millimeters. This uorescence spectrum outside
the NIR window is a disadvantage for surgical application, but
it has minimum toxicity and 5-ALA is naturally present in the
body (Figs.1.6 and 1.7).
Approved in 2017 by FDA, it is used mainly for FGS for
guided ablation of malignant glioblastoma-based. In addition,
5 ALA has also been used in clinical settings for the photodynamic detection and photodynamic therapy of many supercial
skin lesions, and its use is being investigated in several cancer
types [4, 38].
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The main limit of extrinsic probes is the limited approved
number for clinical use. The approval process is long, because a
good uorescent probe should have good uorescent properties,
low toxicity, great quantum yield, and short half-life. Indeed,
depending on the uorophore uptake from the target tissue, the
greater the quantum yield is, the smaller the dose to achieve effectiveness. Moreover, short half-life implicates rapid clearance so
the possibility to repeat uorophore administration for further
intraoperative evaluation.
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800
Wavelength (nm)
Intensity
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1.0
0.8
0.6
0.4
0.2
0
200400 600
Fig. 1.7 Absorbance and uorescence-emission spectra of Protoporphyrin
IX. Continuous and dotted lines show absorbance and emission spectra
respectively
L. Baldari et al.
Extrinsic Probes Under Clinical Development
Even if the approval process is long and difcult, there is high interest in uorophore development for clinical use. A recent review
reported that a total of 85 trial have been registered to test 39 new
contrast agents for uorescence-guided surgery. Some of these
probes under development utilize target like antibodies or peptides
to obtain highly specic uorescent signal. For example, optically
active probes can specically label intra- and extracellular biomarkers of cancer. Targeted uorophores actually under development
aim mainly to identify cancer cells in order to diagnose tumor early
and to verify that tumor dissection is complete. Other untargeted
probes generate uorescent signal through enzymatic process.
Others have uorescent properties themselves [4, 39].
Some of these probes for uorescence-guided surgery have
reached phase III clinical trials:
• SGM-101. It is a target antibody for carcinoembryonic antigen
(CEA) linked with a NIR uorophores. It has been tested in
colorectal tumors, showing high specicity in primary tumor
tissue and in metastases [40].
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• BLZ-100. It is a chlorotoxin peptide linked to ICG, with
afnity for metalloprotease 2. It has been studied for pediatric central nervous system tumor, showing specicity for
tumor tissue and correlation with uorophore dose and cancer grade [41].
• OTL38. It is composed of folic acid and NIR uorophore
S0456. It has demonstrated high sensitivity for ovarian cancer
tissue [42].
Several other probes under development are in phase II or
phase I clinical trials. Some of these contrast agents are BBNIRDye800CW for brain cancer, uorescein-conjugated Wisteria
oribunda for colon cancer, and EC17 for renal carcinoma.
Clinically approved uorescence imaging systems are com-
patible with FDA-approved contrast agents. Thus, most of the
systems are set for ICG, with excitation and emission wavelength around 800nm. Few imaging systems have capability
for uorescein and 5-ALA uorescence, and only two systems
have the possibility to perform uorescence surgery with
methylene blue in the spectrum of 700nm. As a result, because
uorescence is centered around 800nm, the most used uorophore for contrast agents under development is the
IRDye800CW.Thus, there is a gap between uorescent properties of new contrast agents and imaging possibility with
approved imaging systems [4].
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Clinical Imaging Systems
Fluorophores with excitation and emission spectra in the nearinfrared wavelength range (700–900nm) have attracted the most
attention owing to their improved depth penetration range compared with uorophores that emit electromagnetic radiation of
shorter wavelengths. Within this NIR window, the absorption of
most biomolecules (i.e., deoxyhemoglobin, oxyhemoglobin,
water, and lipid) reaches minimal levels, and scattering and autouorescence are relatively low. Since NIR uorescent light is
essentially invisible to the human eye, special imaging systems
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are required to excite the NIR uorophores within the surgical
eld and to collect emitted photons. Well-designed NIR uorophores are needed to highlight the specic structures desired by
the surgeon. The commercial development of these agents for
clinical application has synergized with concurrent improvements in detection instrumentation and software [1].
ICG becomes uorescent once excited with NIR light or with
a dedicated laser beam. The uorescence can be detected using
specic scopes and cameras and then transmitted to a standard
monitor allowing identication of anatomical structures where
the dye is present (i.e., biliary ducts, vessels, lymph nodes, etc.)
[43] (Fig.1.8).
ICG is the only NIR uorophore employed to date for human
use. As described previously, it had widespread uses in hepatic,
cardiac, and ophthalmologic studies, and its use is recently reported
in analyzing tissue perfusion and identifying lymph nodes in cancer patients. It has several clinically excellent properties, which
has been thoroughly veried during its long clinical use [36]:
• Patient safety (nontoxic, nonionizing).
• Ideal for angiography because it binds efciently to blood lipo-
proteins and does not leak from circulation; ideal for bile duct
study because it is excreted selectively through the bile.
• Short lifetime in blood circulation allowing repeated applica-
tions.
• Good signal-to-background ratio (SBR): there is not much
NIR autouorescence in tissue where the exogenous dye is not
present.
• Deep imaging with possibility to see beyond the surface, at a
depth of several millimeters.
• Simple and relatively cheap imaging devices.
L. Baldari et al.
Components ofFluorescence-Based
Surgical System
Fluorescence technology used in surgical system is based on
methodology already dened in uorescence microscopy and
spectroscopy [44]. The system is made of an excitation source, a
Fluorophore
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filter > 760 nm
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Light
Light
source
source
800 nm
760 nm
CCD
Camera
Fig. 1.8 Operating principle of Fluorescence Guided Surgery (FGS)
uorescent probe (described above), lters, and uorescent
detector.
Excitation Source
The excitation source to obtain a uorescent signal is a light
that can be generated by xenon lamps, light emitting diodes
(LEDs), and laser diodes. Even if lamps are exible, allowing a
wide range of wavelength, they are not the ideal source as they
produce heat, requiring a warm-up period, and deteriorate with
use with a consequent decrease of brightness. LEDs are more
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diffused. If compared to xenon lamps, LEDs have longer lifetimes, do not need warm up period, have lower consumption,
and have a narrow bandwidth. Laser diodes have a narrower
wavelength and higher intensity compared with the other two
sources. Laser source is more expensive than LED ones, so they
are less diffused [4].
L. Baldari et al.
Fluorescence System Filters
Excitation and emission lters are necessary to select wavelengths and unwanted signal. For example, emission lters can
be used to narrow the uorescent signal collected to the spectrum
of interest. Emission lters’ features should be clearly dened
and balanced, as a large band allows higher intensity of the signal
but less specicity resulting in lower contrast between the target
and the background. Moreover, the lter has to be chosen according to the Stokes shift of the uorophore, that is the difference (in
wavelength or frequency units) between positions of the band
maxima of the absorption and emission spectra of the same electronic transition [4].
Fluorescence System Detectors
The aim of the detector is to quantify the uorescence of the
single photons according to quantum efciency, that is the ability to convert the incident light to excited electron, and internal
gain, that is the ability to amplify the signal in a large electrical
signal.
To date, several systems are currently used to detect photons in
surgical eld:
• Photomultiplier tubes absorb the incident photons and produce
electrons to obtain the signal.
• Microchannel plate photomultipliers absorb the incident pho-
tons and produce electron with an amplication of the signal
and improved time resolution of the detection system.
• Charge-coupled devices are made of pixel array that produce a
signal proportional to exposure time of incident light, resulting
in high sensitivity.
• Single photon avalanche photodiode generates current quickly
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after photon absorption, thanks to a multiplexed pixel array.
The main advantage is the higher quantum efciency.
Each of these detectors has advantages and disadvantages, and
even if single photon avalanche photodiode has high potential for
uorescence lifetime use, photomultiplier tubes and chargecoupled devices are still currently used [4].
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Currently Available Devices forFluorescenceGuided Surgery
Nowadays, a growing number of companies are developing new
systems for FGS.These systems that are suitable for both nearinfrared (NIR) uorescence and white-light (WL) imaging, integrating the aforementioned technology, available for both open
and minimally invasive surgery.
Each system differs from others on some key features:
• The “exciting” light source type (neon light, LED, or laser
beam).
• The system of signal detection.
• The wavelength emitted and captured.
• The optimal distance to visualize uorescence signal.
• The strength of SBR.
• The possibility of directly overlaying the NIR images to the
WL ones.
The different features that characterize each device (eld of
vision, zoom capability, type of light source, NIR wavelength
emitted and captured, etc.) will have an impact on system performance during surgery. During surgical procedures, the alternate
exposure from WL to NIR light (ICG mode) is used to identify
anatomical structures, blood perfusion, and other details. The uorescence imaging systems allow to obtain uorescent images in
real-time setting. They are quite unexpensive, if compared to some
other technological equipment widely adopted in surgery [45].
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