Hindawi Publishing Corporation
International Journal of Biomedical Imaging
Volume 2012, Article ID 940585, 26 pages
Jarmo T.Alander,1Ilkka Kaartinen,2Aki Laakso,3Tommi P¨ atil¨ a,4
Thomas Spillmann,5ValeryV.Tuchin,6,7,8MaaritVenermo,9andPetri V¨ alisuo1
1Department of Electrical Engineering and Energy Technology, University of Vaasa, Vaasa, Finland
2Department of Hand Surgery, Tampere University Hospital, 33680 Tampere, Finland
3Department of Neurosurgery, Helsinki University Central Hospital (HUCH), Helsinki, Finland
4Department of Cardiosurgery, Helsinki University Central Hospital, Helsinki, Finland
5Department of Equine and Small Animal Medicine, University of Helsinki, Helsinki, Finland
6Saratov State University, Saratov 410012, Russia
7Institute of Precise Mechanics and Control, Russian Academy of Sciences, Saratov 410028, Russia
8University of Oulu, Oulu, Finland
9Clinic of Angiosurgery, Helsinki University Central Hospital, Helsinki, Finland
Correspondence should be addressed to Jarmo T. Alander, firstname.lastname@example.org
Received 1 September 2011; Accepted 1 February 2012
Academic Editor: Guowei Wei
Copyright © 2012 Jarmo T. Alander et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluores-
cence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in
Fluorescence Imaging (FI) is one of the most popular im-
aging modes in biomedical sciences for the visualisation of
cells and tissues both in vitro and in vivo . The benefits of
wavelengths are used for illumination and recording
(cf. Figure 4);
(ii) high sensitivity: extremely small concentrations can
often be made visible;
(iii) Gives molecular information: makes some (bio)
chemistry spatially and temporally visible;
(iv) great tools for research: several possible imaging
modes, most of which are unique;
(v) cheap: the optical instrumentation and computing
needed are quite simple;
(vi) easy to use: resembles classical staining.
Fluorescent imaging is a relatively recent imaging meth-
od and thus still developing in many ways. This is especially
true for indocyanine green (ICG) imaging in its new clinical
applications recently proposed in various branches of sur-
gical medicine, although it has been used in some clinical
applications routinely already for almost sixty years. Thus,
ICG is well known in its established clinical applications,
which greatly facilitates its introduction to new applications.
ing seems to be among the main areas in which ICG imaging
(ICGI) has potential for major developments, for example,
for analysis of ICG fluorescence dynamics  (cf. Figure 2).
This means, among other things, that a lot of computing
development work is still needed for a broader acceptance of
various emerging ICG-based medical imaging methods .
1.1. Indocyanine Green Angiography. Indocyanine green has
been used for decades in ophthalmology for imaging retinal
blood vessels, that is, in retinal angiography. However, fluo-
rescein operating in visual wavelengths has been much more
2International Journal of Biomedical Imaging
Figure 1: A typical ICGA image: heart of a rat. Coronary arteries
clearly visible. Liver shining on the right. Magnification 20×. Image
taken by Dr. Outi Villet at HUCH by our prototype microscope
device shown in Figure 6.
popular in retinal angiography partly because it is visible
without any electronic cameras. However, the objects of
imaging, retinal layers, with fluorescein and ICG somewhat
differ. ICG gives information about deeper lying blood veins
because it operates in near infrared (NIR), in which tissues
are much more translucent than in visual wavelengths.
The principle of fluorescence imaging used in ICG
angiography (ICGA) is simple: illuminate the tissue of
interest with light at the excitation wavelength (about 750 to
800nm) while observing it at longer emission wavelengths
(over 800nm; Figure 4). To create a simple ICGA device,
only a couple of filters are needed in addition to a proper
camera and a light source, which can be quite small and
suitable even for portable use . The filters are needed to
prevent the mixing of the excitation (strong) and fluorescing
(weak) rays to sum at the sensor. Even if the fluorescence is
only a small fraction of the excitation intensity (Table 9: row
1 versus row 10), a surprisingly good signal to noise ratio
(SNR) is attained: a brightly fluorescing object, mostly blood
vessels containing ICG, can be clearly seen on an almost
black background (see Figure 1). Without the filters, the
weak fluorescence image cannot be seen among the strong
reflection of the excitation light.
Indocyanine green dye was developed for near-infrared
(NIR) photography by the Kodak Research Laboratories in
1955 and was approved for clinical use already in 1956 [6, 7].
However, it took over ten years before ICG was used for
angiography . For retinal angiography it has been used
from early 70s .
1.2. Related Work. A few reviews of ICG and ICGA have
been published. Those are briefly reviewed in what follows.
Frangioni gives a review on in vivo fluorescent imaging
including ICG-assisted imaging . Amiot et al. give a
review of the different NIR fluorescent materials developed
a review of the toxicity of organic fluorophores including
ICG used in molecular imaging . For a recent review
of ICG in retinal angiography, see for example, [13–15].
ICG, and infracyanine green in macular hole surgery are
Table 1: The number of ICG-related publications: queries from
databases PubMed, ISI, SPIE, and IEEE (26.7.2011).
ICG and “surgery”
ICG and “liver”
ICG and “retina”
ICG and “cancer”
ICG and “tomography”
ICG and “imaging”
ICG and “heart”
ICG and “wound”
ICG and “lymph”
ICG and “brain”
ICG and “breast”
ICG and “laparoscopy”
reviewed in . ICG and some similar dyes in vitreoretinal
surgery are reviewed in . A short overview of early works
is given in . A recent review of ICG in assessment of
liver function is given in , and a personal history view
on ICG in liver monitoring is given by Paumgartner .
Houston gives an overview of in vivo small animal studies on
fluorescent contrast agents . te Velde et al. have recently
briefly reviewed all papers regarding fluorescent dyes in
oncologic surgery , Polom et al. ICG usage in oncology
and especially in sentinel lymph node biopsy (SLNB) 
imaging . In a recent review Kaiser et al. review optical
methods, including ICG imaging, in noninvasive assessment
of burn wound severity . An excellent and illustrative
review of ICG in clinical imaging of the lymphatic system
is given recently by Marshall et al. . National Library
of Medicine maintains a database of contrast agents called
1.3. ICG Publications. To get an idea of the volume of ICG-
related research activities, the number of ICG-related publi-
cations in several databases (PubMed, ISI, IEEE, and SPIE)
was collected in Table 1 and classified according to the
main application areas. As anticipated, most research on
ICG seems to be related to clinical sciences and not to,
for example, engineering, optics, spectroscopy, or imaging,
which indicates that there is still much work left to reveal
all the technical potential of ICG. For instance, most of the
On the other hand, the long and routine use of ICG in some
clinical applications, such as retinal imaging, has provided
us with much invaluable knowledge and experience useful
in the development of new clinical applications, which are
anticipated to be introduced exceptionally swiftly and at the
same time at both relatively low risk and cost.
International Journal of Biomedical Imaging3
(a)(b) (c) (d)
deep lying arteries in red, (b) at about 60s showing mainly capillaries in yellowish green, (c) at about 90s showing mainly subcutaneous
veins in blue, and (d) fusion of the first three images. Image processing steps: negative of the original image and some intensity remapping.
HUCH from Tokyo Medical and Dental University Hospital of Medicine) and later processed by one of the authors (P. V¨ alisuo).
The number of annual ICG publications according to
PubMed is given in Table 2. The increase of publications has
been about 10 papers per year. This number is expected to
increase due to the emerging clinical applications described
later in this paper. We can already (Fall 2011) see a
considerable increase of papers for the years 2009 and 2010.
According to Espacenet (6.5.2010), there are over 170 ICG-
The principal advantages causing the rapid acceptance of
ICG were the presence of the absorption maximum, around
800nm, the confinement to the vascular compartment
of 50–80mg/kg for animals http://www.drugs.com/pro/
indocyanine-green.html), and the rapid excretion, almost
exclusively into the bile.
ICG fluoresces at about 800nm and longer wavelengths.
The exact shape of the spectra depends somewhat on the
chemical environment and physical condition of ICG mol-
ecules like temperature and ICG concentration. The spectra
are also smoothly varying, thus the exact wavelength values
given in the literature somewhat vary depending also on
the excitation light spectra and the filters used. Table 5
gives some excitation and observation wavelengths used
in different ICG imaging instruments. The sensitivity of
fluorescence spectra on molecular environment means that
ICG is a potential molecular probe . This has not yet
been used in clinical applications. This is obviously one
potential direction of ICG imaging development. Related to
this direction is the need for better understanding of the
Table 2: From PubMed (2.11.2009) “Indocyanine”: (years 2007–
Year NumberYear Number
binding of ICG molecules in different cells and tissues. This
is clearly an arena for some further systematic basic research
using fluorescence microscopy that may later possibly even
lead to some major imaging innovations in biomedical
4International Journal of Biomedical Imaging
ICG has several clinically excellent properties, which has
been thoroughly verified during its long clinical use:
(i) patient safety: nontoxic and nonionizing,
(ii) ideal for angiography: binds efficiently to blood lipo-
proteins, that is, it does not leak from circulation,
(iii) short life time in blood circulation allowing repeated
(iv) good SNR: there is not much NIR autofluorescence
in tissue giving low noise background,
(v) deep imaging: operates in tissue optical window
(vi) simple and cheap imaging devices (Hamamatsu:
What is so new in ICG angiography? Recently new suc-
cessful medical applications, mainly in surgery, have been
introduced. Some of the ICG’s subexcellent properties pro-
vide further challenges to research and engineering develop-
(i) ICG is very recent in many applications such as
cancer treatment, reconstructive surgery, and even in
(ii) ICG needs some NIR imaging device to be visible,
(iii) for some applications ICG seems to need online illu-
mination control facility,
(iv) clinically usable chemical derivatives for more spe-
cific physicochemical imaging do not yet exist,
(v) ICG injection solution contains some sodium iodide;
thus, an allergic reaction is possible,
(vi) ICG is unstable in solutions (10h) and when exposed
to light, and
The development work for creating even better NIR
contrast agents is going on in a few laboratories. Some of the
proposed new molecules are based on ICG, while there are
also totally different approaches such as quantum dot-based
contrast agents [31, 32].
2.1. Structure and Stability. Indocyanine green is a tricarbo-
cyanine dye having a molecular weight of 751.4Da. It is a
negatively charged ion that belongs to the large family of
cyanine dyes . Dry ICG is stable at room temperature.
This is also the form of pharmaceutically available ICG. ICG
is soluble in water (1mg/mL) but is not readily soluble in
saline. Therefore, ICG should first be dissolved in water and
only after this diluted with saline if an isotonic solution is
needed (Sigma-Aldrich). Some chemicals, such as sodium
polyaspartate (PASP), can be used to stabilise ICG in water
and blood solutions, for example, when blood samples
should be stored for several days. The use of PASP has
been demonstrated also in vivo for a rat model [34, 35].
The chemical decomposition of ICG can be inhibited by
sodium azide NaN3, a quencher of singlet oxygen, that is,
an antioxidant . Also storage of the ICG solution at low
temperature (4◦C) inhibits decomposition, while storage at
room temperature facilitates decomposition .
2.1.1. Spectral Stability. In aqueoussolutions, ICG molecules
tend to aggregate, which influences their optical properties
. The aggregation depends on concentration and time;
thus, ICG solutions do not follow Lambert-Beer’s law above
15mg/L in plasma . The spectral stabilisation is fastest
when ICG is dissolved in distilled water, and thus Landsman
et al. do not recommend adding isotonic saline and/or
albumin to the injectate, when fast spectral stability is es-
sential, for example, when using ICG for quantitative pur-
poses . In tissues and cells the NIR absorption peak, due
to binding with cell proteins, is moved to longer wavelengths
2.1.2. Photochemical Stability. When excitated ICG is sup-
agent. Engel et al. have recently studied the stability of ICG
when exposed to light and the production or the conse-
quences of singlet oxygen production of ICG . According
to their observations the decomposition of ICG is due
to singlet oxygen, but it seems that the singlet oxygen is
immediately bound to the decomposition products of ICG
itself. Therefore, it seems that ICG is not a very good source
of singlet oxygen. This has two main consequences with
respect to clinical applications: firstly, ICG can be used with-
out much worry of phototoxicity due to singlet oxygen
production; secondly, when ICG is used as a photodynamic
or photothermal agent, its decomposition products may
be the main cause of phototoxicity. The decomposition
products thermally decompose further to several carbonyl
compounds. However, according to a recent study by Tokuda
et al., ICG seems to be somewhat phototoxic for the retina
Engel et al. tested several solvents for light-induced
decomposition of ICG. What is again interesting and en-
couraging for angiography applications is that ICG in blood
plasma was found to decompose so that only a small amount
of decomposition products were recorded when com-
pared to ICG in water. They suggest that the singlet oxy-
gen produced is quenched by some plasma proteins thus
inhibiting ICG decomposition by singlet oxygen . Very
recently Sato et al. studied the effect of broadband light
on ICG toxicity by filtering the long wavelengths focused
on cultured M¨ uller cells. According to their observations
filtering prevents phototoxicity .
2.1.3. Protein Binding and Fluorescence Life-Time. The im-
portant property of fast binding to plasma proteins, espe-
cially lipoproteins, [42–44] makes repeated intraoperational
applications of ICG possible. The binding to plasma proteins
does not seem to alter protein structures, which is one sign
of nontoxicity . It seems that ICG actually binds to the
lipids of lipoprotein complexes (β-lipoprotein ), and
that the bind results in more intense fluorescence than ICG
bound to for example, free cholesterol . Binding to
International Journal of Biomedical Imaging5
Table 3: ICG cytotoxicity studies.
Spin. root ax.
ICG 1mg/mL; not toxic
IfCG; no damage
ditto; some damage
ICG; no damage
IfCG; no damage
Cell cycle arrest and apoptosis
Fluorescent lamp illumination:
IRDye 800CW; no toxicity obs.
Growth inhibition and damage
ICG interactions with RPE
blood proteins also shifts, slowly, taking several minutes, the
absorption peak, at 780nm, towards longer wavelengths, to
805nm . The absorption peak maximum was observed
at 810nm in the epidermal cell cultures , and at 805–
810nm in the human skin in vivo [47, 48]. The emission
peak is also shifted similarly . Not only the shape of the
spectra is influenced by the chemical environment, but also
the fluorescence life-time changes, a fact which can be used
to probe the molecular environment of ICG and similar dyes
2.2. Physiology and Pharmacokinetics. ICG does not have
any known metabolites, and it is fast extracted by the liver
into bile juice. The transport is done by a protein called
glutathione S-transferase without  modification. Caloric
restriction seems to significantly increase the plasma clear-
ance rate at low doses (0.5mg/kg) . The protein spectra
of different liver diseases also affect ICG protein binding
in blood [43, 51]. Reekers et al. provide a recent study of
the plasma disappearance rate for ASA physical status I-II
The typical dye concentrations used for in vivo retinal
and choroidal 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. No significant
toxic effects have been observed in humans with the high
dose of 5mg/kg of body weight . Table 3 gives a brief
overview of toxicity studies done with ICG.
2.3. Penetration. ICG works in the so-called tissue optical
window, that is, the NIR light used both in excitation and
fluorescence penetrates tissue several millimeters or even
further. This translucency helps to observe, for example,
vascular structures that might be buried in clots or dura
[62, 63]. The penetration depth of light energy into skin
and underlying tissues can be calculated on the basis of
in vivo measurements of optical density OD (accounting
tissues defined as OD = log10I0/It, where I0is the measured
back reflected intensity, and Itis that of the reference. Such
measurements done for 12 healthy young subjects at 775,
807, and 827nm gave the following dependencies for a 3mm
layer of subcutaneous fat: OD775 = 3.2; OD807 = 2.4;
OD827 = 1.6 . The low absorption and high scattering
allow for providing smooth and intensive enough indepth
irradiation of skin tissue due to the photon recirculation
2.4. ICG Derivatives and ICG-Like Contrast Agents. While
NIR fluorescence (NIRF) imaging has recognised potential,
only ICG is a clinically approved NIRF dye. Perhaps in the
future there will be a larger set of NIRF dyes. At least work
on developing new NIRF dyes has been going on and has
already introduced several potential NIRF dye candidates.
Here we will only briefly review some recent development
of ICG derivatives.
While ICG is rapidly bounded with lipoproteins in
blood, it is natural to combine ICG with nanoparticles of
doped with ICG [67–70]. Ogawa et al. have conjugated ICG
with several antibodies in order to target ICG to cancer cells.
However, ICG conjugated to protein usually markedly looses
efficiency, the ICG-antibody complex should be dissociated
so that ICG can be used as an in vivo molecular imaging
probe . S. Achilefu’s group has recently conjugated ICG
with folate-polyethylene glycol for tumor targeting .
Ebert et al. have compared the pharmacokinetics of ICG to
its hydrophilic derivative called SIDAG with a mice model
for breast cancer imaging .
Several encapsulations have been implemented with ICG
[74–78]. Makino et al. have labeled lactosome with ICG. The
labeled lactosome was found to be stable in blood circulation
6International Journal of Biomedical Imaging
and gradually accumulated specifically at a mouse model
liver tumor site . Barth et al. have engineered calcium
targeting human breast and pancreatic cancers .
Infracyanine green (IfCG) (Laboratoires SERB, Paris,
France), also known as IFC green, is ICG without iodine. It
is believed that IfCG is less cytotoxic in macular applications
because 5% glucose solution instead of pure water is used as
its solvent. According to , IfCG is less than ten times as
retina cytotoxic as ICG. Infracyanine green was used in the
study of macular pucker surgery . The absorption and
emission spectra of the commercial ICG and IfCG products
in several solvents and concentrations are compared in .
In this section, an overview of ICG imaging from the in-
strumentation engineering point of view will be given. In-
docyanine green imaging belongs to the class of optical flu-
orescence imaging. Correspondingly, when used with an
operational microscope it closely resembles fluorescence mi-
croscopy. Thus, the instrumentation needed is similar or
even exactly the same as that for fluorescent imaging in gen-
eral, or fluorescence microscopy in particular.
is done so that both visible or excitation and fluorescence
images are displayed together as one image. The fluorescence
image alone may contain only a few details so that the visible
image greatly helps to locate the fluorescing parts with the
help of the landmarks seen in the visible image. Typically the
fluorescence channel is shown, rendered, in colors like vivid
green, having a striking colour contrast to the visible image
of tissues. This kind of visualisation is especially important
in intraoperational use, where the fluorescing parts, like
blood veins, should be recognised easily and immediately. In
order to be able to combine the two images, they should be
aligned correctly. This is called image registration, and it is
[83, 84], while the rendering of the two images for display is
a straightforward operation.
However, the image registration problem can be totally
avoided by optical means by using an ordinary beamsplitter,
which is a dichroic mirror splitting and filtering the beam
into two parts: one for the visible camera and the other for
the NIR camera. This means that both cameras see exactly
the same field of view (FOV), and no registration is needed,
provided that the cameras have identical optics and are
located correctly with respect to each other. In addition to
the beamsplitter, suitable exchangeable filters embedded in
the optics or in a separate filter cube, which is the usual
arrangement in fluorescence microscopy, are used in front
of the cameras to block unwanted wavelengths from entering
camera, so that the excitation light does not mix with the
fluorescence signal because both are summed at the sensor
and inseparable in the resulting electronic image. Visible
range cameras usually already contain filters that block most
of the NIR radiation that would otherwise be summed
for source; Fc: low-pass filter (barrier) for camera) and the emission
spectrum of two NIR LEDs having the nominal peak wavelengths
of 780 and 850nm and full width at half maximum (FWHM)
bandwidths correspondingly of 30 and 95nm.
to a varying extent to the different RGB channels of the
visual image, thus distorting its perceived colours. The main
an operational microscope doing ICG fluorescence imaging
with a beam splitter is that the illumination, which is epi-
scopic in the fluorescent microscope and thus done via the
beamsplitter, is replaced by an ordinary colour camera, while
The excitation light should not contain fluorescence wave-
lengths, they should originate only from the fluorescing ICG.
Thus, a filter is needed to block longer wavelengths from the
launched excitation light, when using a broad spectrum light
source. Ideally the two filters should divide the spectrum
into two nonoverlapping bands (Figure 3). This can be best
done using interference filter pairs, which can be tailored
for any wavelength range and which can have a very narrow
transition band. Commercial interference filter pairs are also
available for ICG fluorescence, separating the spectrum at
about 800nm (Chroma Technology, Brattleboro, VT, USA)
(see Figure 3). When using a light source with a narrow
spectrum, a laser, there is no need to use any excitation
light filter. The use of a beamsplitter is a particularly simple
and practical way of solving the otherwise challenging image
registration problem and allows an illustrative blending of
the colour image and the ICGA to be easily done online,
which is often vital for critical intraoperational use (Table 4).
3.2. Example of an ICGA Device Design. In this section, we
will, in principle, design a simple ICGA device. The principle
of fluorescence imaging is given in Figure 4. In order to see
the fluorescence, which has only a fraction of the intensity
of the excitation light, the latter should not contain any
fluorescence wavelengths. If a broadband light source, like
a halogen lamp, is used, there should be a filter to cut the
longer wavelengths (Fs). In the case of using lasers, which
are monochromatic, no filter is usually needed. On the
International Journal of Biomedical Imaging7
Table 4: ICGI instruments. KAIST: Korea Advanced Institute of
Science and Technology.
Florida Int. U.
Osaka Med. Coll.
U. Clinic Munich
Imaging Station FX 
Ditto 4000 MM
Leica OH3 FL800 
Human leg im.
Blood and ICG
Figure 4: The principle of fluorescence imaging. The radiation
from the light source is filtered by a high-pass filter, Fs, to remove
the fluorescent wavelengths. The blood and ICG suspension under
a tissue absorbs the excitation wavelengths and emits in fluorescent
band. The emitted light is received by the sensor through a low-pass
filter, Fc, to remove the excitation light reflected from the source.
the excitation wavelengths, and possibly shorter wavelengths
should be cut off (Fc). As can be seen in Table 5, there is
some freedom when selecting the exact filter wavelengths.
In addition to the filter wavelengths we should also look at
the wavelength dependence of the light sources, filters, and
cameras. In an ideal case no excitation light is recorded by
the camera while as much as possible of the fluorescence is
minimise excitation light leakage while maximising recorded
fluorescence light. What makes this a nontrivial technical
problem is the shape of the spectra of each component
needed and the other properties of the components affecting
recording. For example, the quantum efficiency of the silicon
semiconductor-based image sensors in NIR band typically
Table 5: ICGI instrument properties.∗: Hitachi, λe[nm] emission
wavelength (min), λc [nm] camera wavelength (min), and cCCD
Table 6: Light source properties.
Rather narrow Monochromatic
Not much Some
Rather highHigh (pulses)
Quite fastSlow-very fast
SpecialVisual imaging Small size
No filtering needed
Filter neededSpeckle pattern
New tech.White light needed
strongly decreases when the wavelength increases. The quan-
tum efficiency means the fraction of photons striking the
sensor that are actually recorded. For silicon sensors, it is
in the visible wavelengths typically around 70%, while for
down to 10% or even less (Figure 5).
3.2.1. Light Sources. Table 6 describes the basic properties
of light sources available for fluorescence imaging. As we
have seen in Table 5, all the basic light source types have
been used in some of the existing ICGA implementations.
Most frequently LEDs and halogen lamps have been used for
illumination. In some experiments also lasers, mainly semi-
conductor diode lasers, resembling LEDs, have been used. In
this example, we will look closer at LEDs. LED light is not
totally monochromatic but contains wavelengths typically
having a bell-shaped spectrum (Figure 3), which should
not overlap too much with the camera filter spectrum (Fc)
8International Journal of Biomedical Imaging
Table 7: Some commercial NIR camera sensors.
Table 8: The most important pixel parameters of the above NIR
If a visual image is recorded or observed, we naturally
need a white light source. Note, that most microscope lights
filter out NIR wavelengths at least partly.
(peak) wavelength of 780nm (LED 780-66-60, Roithner La-
sertechnik GmbH, Vienna, Austria). The measurement was
done with an HR4000 Spectrophotometer (Ocean Optics,
Dunedin, FL, USA). As can be seen, the wavelength range
is over one hundred nanometers with 30nm bandwidth
able to record NIR. However, most cameras are prevented
the superimposed NIR image would badly interfere with the
visual image. The most important parameters of the camera
sensors are resolution, signal to noise ratio (SNR), and
resolution of the ADC converter, read noise, dark current,
and quantum well depth of the sensor. These parameters
for some selected sensors are listed in Tables 7 and 8:
MT9P031 and MT9V032 (Aptina Imaging, San Jose, CA,
USA) are typical complementary metal oxide semiconductor
(CMOS) sensors, except that the NIR response of the latter
is enhanced. The machine vision camera, Elphel NC353L
(Elphel Inc. West Valley City, Utah, USA) includes the
MT9P031 sensor. The KAI-11002 (Kodak, New York, USA)
is a typical Charge Coupled Device (CCD) sensor. iXon3 and
Neo sensors (Andor Technology plc, Belfast, North Ireland)
are meant for scientific imaging, where high sensitivity is
Quantum efficiency (%)
500600700 800900 1000
Figure 5: The quantum efficiencies of different sensor technologies
in VIS-NIR range. iXon3 is an electron multiplier CCD, ER-150 LL
is Hamamatsu biomedical CCD sensor, Neo is a scientific CMOS
sensor,MT9V032is a CMOSsensor for surveillance, KAI-11002is a
needed. The iXon3 is based on Electron Multiplier CCD
(EM-CCD) technology, whereas Neo is based on the scien-
tific CMOS (sCMOS) sensor. The FL-280 and ER-150 are
corresponding sCMOS and CCD sensors from Hamamatsu
(Hamamatsu Photonics K.K, Shizuoka, Japan).
While practically all silicon-based cameras are somewhat
sensitive to near-infrared, when they do not have a filter to
block NIR wavelengths, unfortunately the quantum efficien-
cy tends to decrease quite rapidly by increasing wavelength
(Figure 5). This decrease of quantum efficiency is an es-
sential issue when designing ICG imaging because the fluo-
rescence peak is quite broad and extends far beyond 800nm,
where the efficiency is quite low. Therefore, the nominal
wavelengths of the light source and filters should be as
short as possible, which is in contradiction with the good
separation of excitation and fluorescence and the fact that
the absorption maximum of ICG should be close to the
nominal wavelength of the excitation light source. The quan-
tum efficiency of ICG is quite low, about 0.3% in water
and 1.2% in blood . This constrains camera sensitivity
especially in video applications. Cooled CCDs are often used
to increase the signal to noise ratio (cf. Table 5). Some-
times image intensifiers (night vision) are used to increase
sensitivity for video recording and to allow low doses of ICG
(microdosing) [21, 27, 105, 106].
3.2.3. Filter Design. For the LED source of Figure 3 we need
a filter that effectively blocks all wavelengths longer than
be found from Chroma (Chroma Technology, Brattleboro,
VT, USA), specially tuned to ICG fluorescence. The filter
International Journal of Biomedical Imaging9
Table 9: An example of the light attenuation in an ICG imaging
Fs, ET775 50x
Fc, ET845 55m
Response of the Hamamatsu ER-150 low light
Power per pixel
Photons per pixel
Signal to noise
Ef = 0.10W/m2
Np= 6.1 ·105photons/s
Ne= 1.5 ·105electrons/s
t = 98ms
SNR = 63dB
HQ845/55m, which is of the interference type, has quite
a sharp pass band between 820 and 870nm, and when
comparing the spectra of the LED (LED: 780, 66, and 60)
we are using and this filter we can see that their spectra
somewhat overlap (Figure 3).
A rigorous approach to ICGA system design would in-
clude numerical analysis of the spectra of the light source,
filters, and the camera in order to find the optimal nominal
wavelength of the components. However, in this study we
have simply resorted to those components that were easily
available and which seemed to fit with each other well
Photometric formulas can be still used in developing
practical rules of thumb to estimate the effect of different
components of the ICG imaging system as follows.
The radiant flux of illumination source (cf. Figure 4), Φ0
[W], distributed over a solid emission angle Ω0[sr], forms
the radiant intensity of I0= Φ0/Ω0[W/sr] (Table 9; row 1).
I0travels through the excitation filter, Fs, the transmittance
of which is TFs[unitless], attenuating to Is= I0TFs, (Table 9;
Et [W/m2], depends on the distance, R [m], and angle of
the incidence, θ, as follows: Et = IsΩ0cos(θ)/R2. The tissue
attenuates the incident irradiance by the factor of Tt. For
1mm of aorta tissue, the Tt ≈ 0.45 [1/cm]  (Table 9;
Part of the incident irradiance is absorbed by blood
and ICG and part of it will be diffusely reflected due to
the scattering of the red blood cells (RBCs). The intensities
of the excitation and fluorescence fields can be calculated
using diffusion theory [108–110]. The scattering coefficient
of red blood cells and absorption coefficients of hemoglobin,
needed in the diffusion model, are listed in [111, 112].
Correspondingly, the absorption properties of ICG are list-
ed in . According to diffusion theory, the diffuse re-
flectance, excluding the fluorescence, is Rd ≈ 0.15. When
the fluorescence is included, the total reflected and emitted
intensity Rt is slightly higher. The intensity of the fluores-
cence emission is Ef = (Rt− Rd)Ee ≈ 0.0027Ee. Increasing
example, when CICG= 6.5 μM/L (0.31mg/kg). The intensity
of fluorescence is nonlinear. It approximately doubles when
the ICG concentration is increased ten fold, that is, CICG =
65 μM/L (3.1mg/kg).
which again attenuates the irradiance by the factor of Tt.
The remaining irradiance is scattered from the surface of the
emitted from the tissue, It, can be obtained from the
irradiance of the skin, EF,t, as follows: It = EF,t/π (Table 9;
uated by the emission filter, Fc. Approximately TFc= 30% of
the energy of the ICG fluorescent spectrum goes through the
filter. Therefore, the intensity of light Icentering the camera
optics is Ic = ItTFc. (Table 9; row 8). The performance of
the optics is often expressed as the so called f -number. The
radiance in the image plane, Eb, is obtained from the radiant
intensity using the f-number of the optics, as follows: Eb=
It/f2(Table 9; row 9).
As we have seen, only a small fraction of the initial
light intensity induces fluorescence which finally will reach
the image plane. To compensate the low light intensity, the
exposure time, t [s], must be relatively long, which increases
the signal level, S = ΦsEbt, where, Φs, is the quantum
efficiency of the sensor. Long exposure time also increases
the level of thermal noise, Nth[e/pixel], due to dark current,
id [e/pixel/s]. The total thermal noise within the exposure
interval, Nth = idt, dominates the total noise, Nr [e/pixel],
when t > Nr/t. Above this limit, increasing the exposure time
increases the total noise level, Ntot= Nr+Nth, approximately
ratio, SNR = S/Ntot, is not significantly improved any more.
Therefore, the light entering the image plane should have
sufficient intensity to keep the exposure time short enough.
The optimal exposure time to can be determined, if
the physical pixel area, Ap, and the maximum number of
electrons the pixel can hold, the quantum well depth, DQW,
are known. The number of photons hitting the pixel is
EbAp/Ep, where Ep = ch/λ is the energy of the photon,
where c is the speed of light, h is Plank’s constant, and λ is
the wavelength of the photon. Therefore, the time which is
needed to fill the quantum well is
As an example, the calculation of the observed fluo-
rescent intensity and the performance of the Hamamatsu
ER-150 sensor is estimated in Table 9 (rows 10–14). Many
parameters used in the calculation are only estimations, es-
pecially the tissue properties above blood layer, the hae-
moglobin concentration in blood, the concentration of the
ICG and the quantum efficiency of ICG. Furthermore, the
fact that the emission spectrum of ICG may depend on
10 International Journal of Biomedical Imaging
Table 10: The loss factors and corresponding attenuations [dB] of
the top five loss factors in ICGA imaging.
The fluorescence of ICG in blood
Losses in the tissue above the blood vessel
Quantum efficiency of sensor Φs
Transmittance of the emission filter
Diffusion losses in the lambertian surface
Other factors together
the spectrum of the illumination is neglected. Therefore,
the absolute values given in the above calculations are not
accurate. However, they provide insight into the losses in
the imaging system. Eventually only about 35ppm (parts per
million) of the original radiation intensity remains in the
image plane, and only about 25 percent of it will be detected.
The summary of top five loss factors causing −48.7dB
attenuation of the total −50.5dB attenuation in the example
system is shown in Table 10. Remember that one full stop
used in camera lenses is equivalent to 3dB. Thus, the total
attenuation corresponds to about 17 full stops, which means
that we need to use large aperture, long exposure time, and
strong illumination in order to get high quality, that is, high
SNR ICG fluorescence images.
3.2.4. Test and Background Light. As NIR light is not visible
to the human eye and fresh ICG-water solution is not always
at hand, it is practical to have a test light to see if the camera
system is working on the ICG fluorescence wavelengths.
We have used an LED SFH485-P (Osram/Siemens, Berlin,
Germany), having peak emission at 880nm, as a test light
to see if the camera is tuned to wavelengths ranging from
about 800nm to 900nm. We have also constructed a simple
light control for using this LED as a background light for
ICG fluorescence imaging. The test light can also be used
as tunable backlight, when we want to see landmarks not
3.2.5. Optics. Our example system was based on an old
operational microscope originally not at all designed for NIR
imaging (Wild, Figure 6). It has two oculars and a C mount
for a camera for both eyes. This gives us an opportunity not
only to record ICGA videos but also ICGA stereo videos.
It has been shown that stereo videos are beneficial in the
training of surgery students .
Excluding the microscope, the cost of our prototype
components including two interference filters, two cameras,
an LED light, and a PC with some software is about 3000
euros. Figure 1 shows a typical image taken by our prototype
system. As can be seen the quality of the image is quite
good, especially when we remember that the optics used (the
microscope) is not designed for NIR imaging. The use of
special NIR optics would considerably increase the cost of
Figure 6: An old operational microscope used in our prototype
ICG stereo video angiography system experiment. Hamamatsu NIR
camera on the left camera arm.
the system while probably only somewhat increasing the
3.2.6. ICGA Test. After technical laboratory tests, our device
was tested by recording ICGA of rat heart (Figure 4). Neither
the use of ICG dye nor the modest optics not originally
designed for NIR imaging does too much restrict resolution,
when compared to imaging in visible wavelengths: arteries,
the caliper of which are only a fraction of millimeter, can be
Using two cameras, stereo images and stereo video can
be taken. The cameras can be attached to an operational
microscope (Figure 6) or simply attached together when
microscopy is not used. This gives literally new vision for
complex scenes can be seen stereoscopically.
Established medical applications of ICG are retinal angiog-
raphy, liver clearance test, and cardiac output monitoring.
ICG is fast removed from circulation by the liver into bile
juice, which is applied in liver condition monitoring. It
also gives the option to inject ICG several times during an
operation if needed. Recent interest in ICG is based on
new applications in surgery and especially in angiography
related to intraoperative monitoring of blood circulation
in vital organs, where intraoperative angiography is also
economically motivated .
4.1. Intraoperational Angiography. As compared to other an-
giography methods (X-ray, CT, MRI, and PET), ICGA can
be easily and economically used intraoperationally, when
blood vessels are exposed allowing direct visual observation,
for example, in neurosurgery, bypass coronary surgery, flap
operations in reconstructive surgery, wound and trauma
surgery, and laparoscopic surgery, where it is vital to check
that blood circulation is recovered properly.
The imaging protocol is simple, and devices are relatively
cheap. ICG is given as an injection (bolus) into systemic
blood circulation and imaging is done during a period of few
International Journal of Biomedical Imaging11
minutes after injection. Normally a new bolus can be given
after about 15 minutes.
4.2. Neurosurgery. Neurosurgery is ideal for ICGA because
operations are already done under a microscope (and cam-
aremainly exposed and thus canbe seen more or less directly
by visual means. Milestones in neurosurgery include
(i) 2001: experiment with surgical microscope (OPMI)
in neurosurgery ;
(ii) 2002: FDA approval for cerebral angiography re-
(iii) 2003: ICGA was introduced for clinical neurosurgery
(iv) 2005: ICGA done with surgical microscope ;
(v) Leica 2006: FDA approval of ICGA surgical micro-
(vi) Zeiss 2007: commercial surgical microscope with
(vii) Zeiss 2009: ICGA dynamics display software.
Earlier, ICG has been used in neurology, for example,
for measurement of cerebral blood flow in newborn infants
Neurosurgical vascular operations are usually performed
to exclude vascular malformations from the circulation or to
provide revascularisation in case of compromised cerebral
perfusion. Typical vascular anomalies to be treated surgi-
cally are cerebral aneurysms and intracranial or intraspinal
arteriovenous malformations (AVMs) and fistulas. It is of
in question has been completely obliterated and removed
from the circulation, and just as critical is to ensure that
physiological blood flow in associated and adjacent vessels
remains uncompromised at the end of the procedure. In
revascularisation, that is, bypass procedures, the patency
of the vascular microanastomosis is likewise paramount to
successful procedures. Incomplete obliteration of a rupture-
prone aneurysm or AVM may result in a hemorrhage, and
occlusion of a parent vessel or an anastomosis in an ischemic
stroke; both of which may have catastrophic consequences
for the patient. Postoperative angiography is useful in
assessing the residual filling of the treated lesion, but in case
of inadvertent vessel occlusion the result of postoperative
imaging comes too late, and the ischemic brain or medullary
lesion has already irreversibly occurred. Although it is pos-
sible to use intraoperative digital subtraction angiography
(DSA) in the operating room, the setup takes a relatively
long time, and thus DSA cannot be used routinely in every
operation. Moreover, DSA is associated with a complication
rate of up to 3%, and its resolution is insufficient to
demonstrate the occlusion of small (<1mm) perforating
arteries, which, despite their small caliber, may supply blood
flow to critical neural structures in, for example, basal gan-
glia and the brain stem. In neurosurgery all complex
operations are performed under high magnification of a
surgical microscope, which provides an excellent hardware
platform for implementing new optical solutions and to
mount various external devices, such as video cameras.
ICG angiography was introduced to neurosurgery in
2003  and has become a routine method for intraop-
erative evaluation of intracranial blood circulation. It has
been used at the Department of Neurosurgery at Helsinki
University Central Hospital since 2005 in approximately
300 operations every year. It provides real-time information
about the patency of vessels of all sizes seen in the field of the
surgical microscope. Its usefulness in intracranial aneurysm
surgery has been recently assessed in several large patient
series, in altogether 620 aneurysms [116, 118–121]. The
uniform conclusion of all the reports was that the correlation
between ICG angiography and postoperative angiography
has been 90–95%, in terms of aneurysm remnants and vessel
branch stenoses or occlusions. In addition, ICG angiography
has the added advantage of demonstrating small perforating
artery occlusion intraoperatively, enabling the immediate
correction of aneurysm clip placement . However, ICG
angiography may be inadequate in cases of giant, complex,
or deep-sited aneurysms . Atherosclerotic calcifications
also limit its reliability in demonstrating, for example, an-
eurysm neck remnants.
During microneurosurgical treatment of brain or spinal
arteriovenous malformations and dural arteriovenous fistu-
las, the dynamic visualisation of different phases of the blood
flow by ICG angiography is helpful in the identification
and differentiation of feeding arteries, arterialised draining
veins, and normal veins, as well as the fistulous sites, during
intraoperative orientation within the surgical field [87, 122–
often have complex 3D anatomy, and ICG angiography is
only able to visualise vessels on the surface of the illuminated
surgical field. Likewise, ICG angiography cannot, at present,
be considered reliable in assessing possible residual AVMs,
which still requires DSA either intra- or post-operatively.
ICG angiography has also been evaluated and found
rosurgical extracranial-intracranial revascularisation bypass
operations . It was also helpful in identifying the tar-
get recipient artery of sufficient diameter (>1mm) in ex-
tracranial-intracranial bypass procedures performed via very
onstrated to be useful in evaluating the patency of extracra-
nial vertebral artery after surgical transposition and in
localizing vertebral artery within its periosteal sheath dur-
ing surgery of cervical neurinomas . Very recently,
Haga et al. have used ICG-VA for assessment of carotid
endarterectomy . Commercially available semiquanti-
tative dynamic ICG fluorescence analysis system has also
been recently suggested to be able to demonstrate impaired
regional perfusion in patients with cerebral ischemia .
The usefulness of ICG angiography in microneurosurgi-
applications are developed, and more experience is gained.
However, there is still room for technical developments,
for example, in form of rapid and reliable flow dynamics
12 International Journal of Biomedical Imaging
loops, since rapid ICG reinjections generally suffer from
lower contrast due to residual ICG inside the vessels.
ideal for ICGA because they are located, like brain arteries,
on the organ they supply blood to. The major milestones of
ICG in coronary bypass surgery include
(i) 2002: a pig model of coronary bypass angiography
with ICG ;
metering at University of Toronto;
(iii) 2005: FDA approval for ICGA device SPY for coro-
(iv) 2005–2009: GRIIP clinical trial (phase III) at Sunny-
brook Health Sciences Centre.
Coronary artery bypass grafting (CABG) is the most
frequent cardiac operation with annual rates of 400,000 pro-
cedures in the United States and 76,000 in Germany. During
these operations verification of graft patency should be a
key aspect, as immediate intraoperative graft failure occurs
in up to 4% of grafts (8% of patients) . At patient
discharge the graft occlusion rate is 5–20% and up to 30%
at one year after the operation . Intuitively, eliminating
intraoperative graft failure and technical failure should
reduce cardiac mortality and morbidity in the short term
and improve clinical outcome in the long term. Although
conventional angiography remains the gold standard tech-
nique for assessing graft patency, it is rarely available in the
operating room and consequently several other less invasive
approaches have been advocated. The most commonly used
intraoperative method is transit-time flowmetry (TTFM),
which measures the mean flow of the bypass graft and
calculates a pulsatility index of the flow pattern. TTFM is
reliable and sensitive in detecting graft failure, but in several
patients it might lead to unnecessary graft revision .
Near-infrared imaging (NIR) based on the intravascular
ICG dosing has emerged as a novel method for graft patency
assessment. Two main systems have been introduced.
perfusion is assessed by imaging an area of interest around a
coronary vessel. In this imaging method, peak fluorescence
intensity and temporal slope of fluorescence intensity in the
tissue are measured . This imaging method has been
shown to agree with the result of the fluorescent microsphere
imaging, which is the golden standard .
Secondly, a direct imaging of the grafts by visualising
the graft lumen by ICG angiography. In an early study by
Rubens et al., 20 patients were studied by intraoperative
ICG angiography, and one patient (5%) was identified as
needing a graft revision . Taggart et al. investigated 213
grafts with a revision rate of 4 grafts (1.9%) acknowledged
by ICG angiography . Reuthebuch and colleagues
published a graft revision rate of 4 (3.7%) out of 107 patients
. Balacumaraswami et al. assessed the intraoperative
graft patency of 533 conduits in 200 patients. Fluorescence
imaging confirmed technical failure in 8 (1.5%) conduits in
8 (4%) patients, necessitating graft revision . Takahashi
et al. reported a study of intraoperative ICG angiography of
290 grafts, in which four grafts (1.9%) were visualised to
348 coronary bypass grafts were studied by ICG angiograms.
In 4.2% of patients information from the ICG imaging led to
graft revisions that would have otherwise gone unrecognised
Intraoperative graft occlusion in CABG is a consistent
finding affecting up to 5% of grafts. This probably causes
difficulties in both the short and the long term. Detection
of technical problems in the most vital graft, the internal
thoracic artery is of utmost importance. Among the available
techniques for assessing graft patency, intraoperative ICG
angiography seems to provide a sensitive method compared
to the mostly used method of TTFM. In a recently pub-
lished randomised trial, 156 patients were randomised to
go through ICG angiography or TTFM during CABG to
assess graft function intraoperatively. One year after the
operation, 43 out of 312 grafts were occluded (13,8%), with
no difference between the groups. Thus, ICG angiography
seems to provide a novel technique in addition to the more
acknowledged range of methods of intraoperative quality
confirmation in coronary surgery .
4.4. Vascular Surgery. In vascular surgery, ICG fluorescence
imaging has been studied in intraoperative assessment of
graft patency, diagnostics of peripheral arterial occlusive
disease and Raynaud phenomenon (RP) as well as in pre-
dicting wound healing after major amputation and to eval-
uate splanchnic circulation. Also, the usefulness of ICG an-
giography in evaluating angiogenesis in small animal models
and in detecting the vulnerability of atherosclerotic plaque
has been tested. In one study ICG imaging was used in the
treatment of varicose veins with sclerotherapy.
In a preliminary report by Unno et al., 9 patients were
recruited in an intraoperative angiography performed with
PDE. At the end of the operation before wound closure, ICG
was injected in a central intravenous line. ICG dye reached
the leg artery about 30 seconds after the injection. In 8 out of
9 cases, ICG angiography showed good fluorescent signals as
the ICG passed through the graft. In one case no fluorescent
signal was detected and during revision a distal thrombosis
was detected and repaired .
Kang et al. have proposed a perfusion rate model based
on ICG dynamics, which they later apply to human patients
to diagnose peripheral arterial occlusive disease (PAOD)
with VasView . PAOD patients and control subjects with
normal vasculature were evaluated for lower extremity tissue
perfusion using ICG perfusion imaging. The perfusion rates
of the lower extremities with severe PAOD were significantly
lower than those of normal controls. Even in cases of mild
PAOD, the perfusion rates were lower compared to the con-
trol, while the conventional methods failed to detect mild
functional impairment. These results collectively indicated
that ICG perfusion imaging is an effective tool for diagnosis
of PAOD, when compared to the golden standard of ankle-
brachial blood pressure ratio [101, 142].
International Journal of Biomedical Imaging13
In a recent study, Kang et al. tested the use of combined
analysis of multiple parameters, especially onset time and
modified Tmax, which means the time from onset of ICG
fluorescence to Tmax, to diagnose Raynaud phenomenon
(RP).To validate the method, they performed a conventional
thermographic analysis combined with cold challenge and
rewarming along with ICG dynamic imaging and segmental
analysis. A case-control analysis demonstrated that the
segmental pattern of ICG dynamics in both hands was sig-
nificantly different between normal and RP cases, suggesting
the possibility of clinical application of this method for the
reliable diagnosis of Raynaud phenomenon .
In patients with no possibility of revascularization, about
half sustain amputation within one year. To maintain best
possible mobility, amputation should be done as distally as
possible. On the other hand, healing of the amputation
wound should be assessed before the procedure to avoid
wound healing problems, infections, and reamputations.
Zimmermann et al. evaluated the use of ICG fluorescence
angiography at an early postoperative time point to predict
the tissue necrosis at the level of amputation. The perfusion
of amputation stumps was measured with the IC-View-
System. In total 10 patients with critical limb ischemia
and ischemic tissue loss were investigated within 72 hours
after major amputation (above knee and below knee) with
indocyanine green (ICG) fluorescence .
Strategies for neovascularization of ischemic cardiac or
lower extremity tissue has been under intensive research
recently. For example, gene technology has been studied
to achieve therapeutic angiogenesis for peripheral arterial
disease. One major problem in this investigation has been
visualization and quantification of collateral growth in small
animal models. The current gold standard of minimal
invasive determination of blood perfusion within the hind
limb of mice is the laser Doppler perfusion imaging (LDPI).
However, it does not penetrate the entire limb and, thus,
measures relative superficial perfusion rather than collaterals
in muscle layer. Wuestenfeld et al. evaluated the applicability
of the ICG angiography for the determination of hind limb
perfusion in mice and compared it to LDPI. The authors
suggest that ICGA is a potent tool for the quantification of
collateral flow in small animal models and that LDPI shows
unreliable high perfusion in the operated foot after one week
indicating that it measures perfusion in the superficial skin
rather than entire hind limb .
Lipid rich vulnerable plaques are the main cause of acute
ICG is a lipophilic molecule that accumulates at sites of lipid
and inflammation. In animal models, it has been shown that
ICG accumulates in lipid in aortic plaques and helps localise
the atheromas. Furthermore, in human carotid artery spec-
imens it has been demonstrated that ICG colocalised with
lipid-rich atheroma and macrophages. Together these results
for lipid-rich and inflammatory atherosclerotic vessel lesions
ICG fluorescence imaging has also been used to measure
splanchnic blood flow. Leppikangas et al. studied the effects
of levosimendan on systemic and splanchnic circulation
during and after abdominal aortic surgery in a double-
blinded randomized study, in which 10 patients received
levosimendan and 10 patients placebo. The total splanchnic
blood flow was estimated by measuring the indocyanine
green plasma disappearance rate (ICG-PDR) transcuta-
neously. Each patient was connected to an ICG finger clip,
which was connected to a liver function monitor (LiMon).
A 0.25mg/kg dose of ICG was injected through a central
venous line of the pulmonary artery catheter at baseline,
before and during aortic clamping, and postoperatively. Le-
nic perfusion in patients undergoing an elective aortic
aneurysm operation .
Foam sclerotherapy is a widely used treatment for vari-
cose veins. The spreading of the sclerosant is usually visual-
ized by ultrasound. Kikuchi et al. reported the development
of visualized sclerotherapy procedure using PDE. Camera
images were digitized for real-time display and reviewed.
Operating lights were turned off during imaging. ICG was
mixed with polidocanol and air. In total, 35 patients were
treated and studied. In all patients, sclerosant spreading was
seen as excellent, and no side effects from ICG were observed
4.5. Oncology and Sentinel Lymph Node Harvesting. The
pioneering work of Chen et al. using a rat model shows that
ICG injected in cancer tumor can be used in laser assisted
photothermal- and photoimmuno-therapies [149, 150]. The
first clinical trials of this kind of therapy have recently done
successfully by the same group .
Lymph nodes are the initial site for metastases for most
cancers. According to surgical principles, all cancer tissue
within the primary tumour and metastatic lymph nodes
should be removed during the surgical operation in order
to achieve a complete and potentially curative resection. The
sentinel node is the lymph node that receives the first lymph
flow from a malignant tumour, and universally it is the
first station, where a potential dissemination of malignant
disease can be identified . A real problem in cancer
surgery is that the lymph nodes are difficult to harvest dur-
ing operation. Currently radioactive technetium-99m iso-
tope labeling is used to detect lymph nodes. This may
be replaced by ICG NIR imaging. This attractive method
to facilitate the visualisation of lymphatic vessels, sentinel
nodes, and metastatic lymph nodes has been introduced
by Lim and Soter . Here, ICG is injected under the
skin from where it flows via lymph circulation to lymph
nodes revealing them when lit with excitation light [92, 98,
154]. More recently, Kim et al. have used a dual-modality
lymph node mapping to detect sentinel lymph nodes in rats,
combining photoacoustic and fluorescence imaging .
Ito and colleagues utilised sentinel node navigation based
on ICG in patients who were diagnosed to have lung cancer
. They concluded sentinel node navigation using ICG
in lung cancer to be feasible, but some modifications will
be necessary before the method can be clinically applied.
sarcoma and cutaneous metastases of a rectalcarcinoma they
have been treated successfully [157–159]. Crane et al. have
14 International Journal of Biomedical Imaging
very recently used ICG in transcutaneous SLN detection in
of lymphatic pathway involved in the spreading of prostate
Albumin affects ICG fluorescence efficiency. Therefore
in some studies albumin, usually human serum albumin
(HSA), has been mixed with ICG in order to increase the
fluorescence efficiency and thus sensitivity of ICG in SLN
detection. However, a very recent randomised, double-blind
comparison of ICG with and without HSA seems to indicate
that there is no increase in sensitivity at least in the case of
breast cancer .
We believe that the possibility to identify lymphatic
vessels and appropriate lymph nodes in the operating room
during surgery would yield marked benefits in terms of com-
pleteness of surgical resection and perioperative evaluation
of potential dissemination of a malignant and deadly disease.
4.5.1. Lymphography. The lymphatic system is vital for many
physiological processes, including immune reactions, and
the maintenance of body fluid and chemical balances. The
lack of noninvasive methods to monitor lymphatic pumping
dynamics has been perhaps the most important reason for
keeping the role of lymphatics modest in the clinical setting.
Unno et al. have recently shown how ICGI can be used
in a minimally invasive method of monitoring human lym-
phatic pumping with a commercially available device and a
custom-made transparent sphygmomanometer .
4.6. Liver Surgery and Laparoscopy. ICG has been used for
many years as a test for hepatic function and to measure
hepatic blood flow in humans and different animal species
. In these tests, ICG clearance has mainly been assessed
by its blood clearance curve [165, 166]. There are a limited
number of studies evaluating the hepatic blood flow and
liver function by direct ICG clearance using NIRS in healthy
rabbits and rabbits with surgically reduced hepatic blood
flow or experimentally induced liver cirrhosis . The
studies showed that measurement of ICG clearance by NIRS
is promising for the assessment of liver dysfunction and may
have applications in hepatic surgery and transplantation.
Furthermore, the technique reflects the reduced liver blood
flow and perfusion in liver cirrhosis more accurately than
excretion determined by NIRS correlated with the degree
of parenchymal liver dysfunction . A recent study in
humans revealed that the measurement of hepatic ICG
uptake by NIRS could become a valuable tool for assessing
the indication for venous reconstruction in living donor
All studies mentioned show the potentials of NIRS-based
determination of ICG clearance for the assessment of
parenchymal liver function and perfusion. However, the
absorption intensity of the liver after ICG injection by NIRS
was determined in all studies by attaching NIRS sensors
to the liver during laparotomy . Injection of ICG
via portal vein and subsequent imaging can be used to
intraoperationally visualise liver segments and subsegments
Ishizawa et al. have used ICG in a routine liver test
preoperationally, after which a prototype ICG fluorescence
imager was used to detect hepatocellular carcinoma intraop-
erationally during a laparoscopic hepatectomy .
In 2009, two clinical studies revealed that real-time ICG-
fluorescent imaging enabled the highly sensitive identifica-
tion of small, grossly unidentifiable liver cancers. This led
to an enhanced accuracy of operative staging and liver re-
section. Currently, indocyanine green fluorescence imaging
navigation is considered to be a promising tool for clinical
exploration for hepatocellular carcinoma and for routine in-
traoperative imaging during hepatic resection [170, 171].
Sentinel lymph node detection has been one main
application of ICG in laparoscopic studies including early
gastric cancer treatment and gastrectomy [172–177]. Harada
et al. compared conventional and ICG-based laparoscopic
sentinel node mapping for colorectal cancer and conclude
that the latter is superior to the former . Miyashiro
et al. have recently applied a prototype ICG laparoscopic
system  to detect sentinel nodes in gastric cancer
surgery . Jeong et al. have developed a new NOTES
procedure for laparoscopic sentinel lymph node dissection
of the stomach with ICG marking using a pig model .
Note, that CO2pneumoperitoneum used in laparoscopic
operations, which decreases liver blood flow, also increases
ICG half-life .
Laparoscopic Cholecystectomy. While ICG is rapidly excreted
via the bile duct, it is most natural to apply ICG intraop-
erationally to aid bile examination and operations .
Indeed, several groups have recently showed how ICG can
be used to aid both open and laparoscopic cholecystectomy
[184–189]. An earlier work by Ikeda et al. uses ICG for
monitoring primary sclerosing cholangitis .
4.7. Reconstructive Microsurgery. For the past 20 years, the
use of different fasciocutaneous perforator flaps has become
popular in the field of reconstructive plastic surgery. Flaps
that are over 15 × 30cm in size can be raised on a single
perforating artery and its concomitant vein. These flaps have
been used to reconstruct a wide range of different tissue
defects. Even a partial loss of the flap can lead to the total
failure of the reconstruction. In perforator flaps, the perfu-
sion of the most distal parts of the flaps is often problematic.
Recently, several reports have shown the feasibility of ICG
angiography in the intraoperative assessment of flap viability
[191–199]. ICG angiography has also been used to assess the
patency of the microvascular anastomosis intraoperatively,
and the intrinsic transit time of the flap circulation with
promising results [200–202]. In the preoperative planning of
perforator flap reconstructions, ICG angiography has been
design of the flap . After free tissue transfer surgery,
the flap circulation has to be carefully followed. Although
other methods exist for continuous flap monitoring, ICG
angiography may be helpful in the early postoperative
phase for the detection of anastomotic thrombosis, when
flap survival is in doubt [144, 202, 204–207]. In large
axial or random pattern flaps, ICG angiography can be
International Journal of Biomedical Imaging 15
used intraoperatively for deciding the need for a delay
procedure to ensure flap survival [205, 208–210]. In breast
reconstructions, transverse abdominal flaps can be raised
on superficial vessels alone (SIEA-flap) or more commonly
using the deep inferior epigastric perforator flap (DIEP or
TRAM flaps). The choice is often made intraoperatively,
and ICG angiography is found to be a beneficial tool in
the decision making [211–216]. There has also been interest
in using ICG angiography in evaluation of tissue viability,
especially in traumatic degloving wounds and burns. This
evidence is sparse, though, and further research is needed
Intraoperative assessment of flood flow is important
for successful transplantations. Hoffmann et al. have used
laser-assisted ICGA (IC-VIEW) for successful intraoperative
assessment of kidney allograft perfusion in 10 consecutive de
novo renal transplantations . Sawada et al. have recently
used ICGA by PDE for intraoperative assessment of renal
vasculature after revascularisation of a transplanted kidney
. Mizuno and Isaji have used ICG-injected intrabiliary
for donor liver bile duct imaging .
4.8. Other Clinical Applications. In addition to the above
surgical applications, the clinical application of ICG include
such topics as brain imaging and hemodynamics [223, 224],
rheumatoid arthritis [225–227], burns and other trauma
, and muscle perfusion .
can be used with a suitable intense light source, typically a
laser, as an ingredient of a tissue solder like albumin, which
could be one method for making surgery more automatic as
laser-based ophthalmic surgery is already today.
4.8.1. Photodynamic and Photothermal Therapy. When an
ICG molecule is excited, it can further transfer energy to
photodynamic therapy agent. In principle, for example, after
with NIR light could be used to destroy metastatic nodes.
ICG binds easily to tissue even at high concentrations,
and the visual change in colour from green to orange is
manifested by the wavelength shift in reflectance peak. ICG
has been used in vitro laser-assisted fat cell destruction,
which might give a new optics-based procedure for cosmetic
Similarly, ICGA can be used as a light-activated antibac-
terial agent (LAAA), for example, in wound healing ,
or treating chronic rhinosinusitis  with near-infrared
laser illumination (NILI). It was shown recently that the
photodynamic effect can be used for acne treatment [241–
Through intense light (laser) irradiation a number of
new effects can be provided, which lead to more effective
bacteria killing and controllable cell destruction and/or
inhibition of excessive synthesis of sebum in sebocytes, like
the localised photodynamic effect based on the appropriate
concentration of the suitable exogenous dye incorporated
into hair follicle or any other skin appendages. The indocya-
nine green is one of the prospective exogenous dyes for soft
photodynamic treatment (PDT).
4.9. Dyeing. Finally, as ICG is a dye it can be naturally
used for tattooing, labeling, and similar tasks [244–249]. A
relatively early work demonstrates how ICG can be used to
stain caries lesions for the further removal of lesion by a
laser by the help of the high light absorption of ICG at the
excitation wavelength . Recently, Kitai et al. have used
ICG for monitoring of perineal wound contamination in
abdominoperineal resection .
Because hepatocytes handle ICG in the liver, ICG can
be used also to monitor differentiation of mouse embryonic
stem cells into hepatocytes [252, 253].
In this work, we have reviewed well over 200 papers describ-
indocyanine green in clinical, mainly surgical, applications.
Many interesting works had to be omitted simply due to
space limitations. However, it is hoped that we have suc-
ceeded in collecting most of the key publications for giving
an overview of the indocyanine green fluorescence technol-
ogy and its most important emerging clinical application.
Many new clinical applications of ICG and ICG angiog-
raphy are just emerging and more are definitely expected to
appear in the near future; thus, it is obvious that much more
of further engineering research include
(i) image processing of ICG and ICGA information, also
in real-time (video and stereo) [143, 239, 254, 255],
(ii) capillary circulation monitoring and perfusion dy-
namics imaging [256–262],
(iii) combining ICG and other imaging modalities like
visual, CT, MRI, and PET [263–266],
(iv) combining ICG and ICGA with dermal imaging
(v) deeper imaging (optical tomography) [261, 267],
(vi) optical imaging device development (laparoscopy)
and optimisation, hands-free [4, 268, 269],
(vii) development of new derivatives of ICG for more spe-
cific imaging modes,
(viii) increasing the quantum efficiency of ICG by, for
example, metallic nanoparticles [270, 271],
(ix) micro- and nano-encapsulation of ICG for nonan-
giography applications [76, 272],
(x) extraction of spectral information and chemometry
(multispectral imaging) , and
(xi) integration of ICG imaging to robotic-assisted sur-
16International Journal of Biomedical Imaging
In a clinical setting, ICG is a new and unique method in
imaging of the lymphatic circulatory system and thus offers
both challenges and the potential for totally new clinical
Anterolateral thigh flap
American Society of Anesthesiology Patient
Bovine serum albumin
Balanced salt solution
Coronary artery bypass grafting
Charge coupled device (camera)
Carotid end arterectomy
Digital subtraction angiography
Enhanced permeability and retention
(US) Food and drug administration
Fluorescence Life-time imaging microscopy
Field of view FT fluorescence tomography
Human serum albumin
Helsinki University Central Hospital
Independent component analysis
ICG-PDR: ICG plasma disappearance rate
ICG-VA: ICG video angiography
ICGA: ICG angiography
ICGI: ICG imaging
ICG-PDR: ICG plasma disappearance rate
ICU: Intensive care unit
IfCG: Infracyanine green
IREE: Infrared-ray electronic endoscopy
ISPI: In situ photoImmunotherapy
LAAA: Light-activated antimicrobial agents
LDL: Low-density lipoprotein
LDPI: Laser Doppler perfusion imaging
LED: Light-emitting diode
LIFE: Laparoscopic intragastric full-thickness
MICAD: Molecular Imaging and Contrast Agent
MRI: Magnetic resonance imaging
NILI:Near-infrared laser illumination
NIR: Near-infra red
NIRF: NIR fluorescence
NIRS: NIR spectroscopy
NOTES: Natural orifice translumenal endoscopic
OCT: Optical coherence tomography
PAD: Peripheral arterial disease
PAOD: Peripheral arterial occlusive disease
PASP: Sodium polyaspartate
PBS: Phosphate-buffered saline
PDT: Photodynamic therapy
SLNB: Sentinel lymph node biopsy
SNNS: Sentinel node navigation surgery
SNR: Signal-to-noise ratio
TTFM: Transit-time flowmetry.
Red blood cell
Red green blue
Retinal pigment epithelium
Sentinel lymph node
The FIELD NIRce project and especially Professor Paul Gel-
adi at SLU University are acknowledged for both their eco-
nomic and scientific support (J. T. Alander and P. V¨ alis-
uo) of this paper and NIR spectroscopy development in
general. This work is a part of the FIELD NIRce
project which is a subproject of Bothnia-Atlantica. A
preliminary version of this work was presented at the
ICNIR 2009 Conference by one of the authors (J. T.
Alander) . V. Tuchin was supported by grants
CRDF (RUB1-2932-SR-08), RFBR(10-07-00526-a). PHO-
TONICS4LIFE of FP7 (224014), RF Ministry of Edu. and
Sci. (1.4.09, 2.1.1/4989, 126.96.36.199/2950), RF Govern. contracts
(02.740.11.0484, 02.740.11.0770, 02.740.11.0879); FDiPro,
Tekes Program, Finland (3081/31/2010). MD Anders Alb¨ ack
at HUCH is acknowledged for his many helpful comments
on vascular surgery, MD Hiroaki Terasaki vising HUCH
from Tokyo Medical and Dental University Hospital of
Medicine for his great help with angiography by the PDE
equipment, and PhD Outi Villet at HUCH is acknowledged
for her help with ICGA rat coronary imaging.
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