The purpose of this paper is to give an overview of the recent surgical intraoperational applications of indocyanine green fluorescence imaging methods, the basics of the technology, and instrumentation used. Well over 200 papers describing this technique in clinical setting are reviewed. In addition to the surgical applications, other recent medical applications of ICG are briefly examined.
Fluorescence Imaging (FI) is one of the most popular imaging modes in biomedical sciences for the visualisation of cells and tissues both high contrast, that is, signal to noise ratio (SNR): only the target, not background, is visible because separate wavelengths are used for illumination and recording (cf. Figure high sensitivity: extremely small concentrations can often be made visible; Gives molecular information: makes some (bio) chemistry spatially and temporally visible; great tools for research: several possible imaging modes, most of which are unique; cheap: the optical instrumentation and computing needed are quite simple; easy to use: resembles classical staining.
Fluorescent imaging is a relatively recent imaging method 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 surgical 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. From an engineering point of view, image and video processing 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 [
Indocyanine green has been used for decades in ophthalmology for imaging retinal blood vessels, that is, in retinal angiography. However, fluorescein operating in visual wavelengths has been much more 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 800 nm) while observing it at longer emission wavelengths (over 800 nm; Figure
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
Simple image processing and pseudocoloring: ICG-VA frames of a leg (toes up) after the injection of ICG: (a) at about 30 s showing deep lying arteries in red, (b) at about 60 s showing mainly capillaries in yellowish green, (c) at about 90 s 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. Fusion by using CMYC model. For more information see [
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 [
A few reviews of ICG and ICGA have been published. Those are briefly reviewed in what follows. Frangioni gives a review on
To get an idea of the volume of ICG-related research activities, the number of ICG-related publications in several databases (PubMed, ISI, IEEE, and SPIE) was collected in Table
The number of ICG-related publications: queries from databases PubMed, ISI, SPIE, and IEEE (26.7.2011).
PubMed | ISI | SPIE | IEEE | |
---|---|---|---|---|
“Indocyanine” (ICG) | 6069 | 5159 | 301 | 57 |
ICG and “surgery” | 2160 | 1059 | 25 | 4 |
ICG and “liver” | 2031 | 1164 | 18 | 5 |
ICG and “retina” | 1176 | 406 | 7 | 11 |
ICG and “cancer” | 816 | 372 | 49 | 14 |
ICG and “tomography” | 748 | 594 | 29 | 10 |
ICG and “imaging” | 697 | 828 | 130 | 43 |
ICG and “heart” | 483 | 174 | 3 | 0 |
ICG and “wound” | 190 | 53 | 24 | 1 |
ICG and “lymph” | 128 | 115 | 11 | 1 |
ICG and “brain” | 127 | 119 | 16 | 0 |
ICG and “breast” | 105 | 189 | 35 | 10 |
ICG and “laparoscopy” | 47 | 26 | 0 | 0 |
The number of annual ICG publications according to PubMed is given in Table
From PubMed (2.11.2009) “Indocyanine”: (years 2007–2010: 17.8.2011).
Recent | Early | ||
Year | Number | Year | Number |
2010 | 397 | 1970 | 38 |
2009 | 369 | 1969 | 35 |
2008 | 295 | 1968 | 29 |
2007 | 275 | 1967 | 37 |
2006 | 274 | 1966 | 25 |
2005 | 277 | 1965 | 20 |
2004 | 295 | 1964 | 16 |
2003 | 295 | 1963 | 8 |
2002 | 240 | 1962 | 5 |
2001 | 219 | 1961 | 2 |
2000 | 224 | 1960 | 10 |
1999 | 195 | 1959 | 0 |
— | — | 1958 | 1 |
1989 | 96 | 1957 | 0 |
— | — | 1956 | 0 |
1979 | 69 | 1955 | 0 |
— | — |
The principal advantages causing the rapid acceptance of ICG were the presence of the absorption maximum, around 800 nm, the confinement to the vascular compartment through binding with plasma proteins, the low toxicity (LD50 of 50–80 mg/kg for animals
ICG fluoresces at about 800 nm and longer wavelengths. The exact shape of the spectra depends somewhat on the chemical environment and physical condition of ICG molecules 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
ICG has several clinically excellent properties, which has been thoroughly verified during its long clinical use: patient safety: nontoxic and nonionizing, ideal for angiography: binds efficiently to blood lipoproteins, that is, it does not leak from circulation, short life time in blood circulation allowing repeated applications, good SNR: there is not much NIR autofluorescence in tissue giving low noise background, deep imaging: operates in tissue optical window (NIR), and simple and cheap imaging devices (Hamamatsu: [
What is so new in ICG angiography? Recently new successful medical applications, mainly in surgery, have been introduced. Some of the ICG’s subexcellent properties provide further challenges to research and engineering development: ICG is very recent in many applications such as cancer treatment, reconstructive surgery, and even in cholecystectomy, ICG needs some NIR imaging device to be visible, for some applications ICG seems to need online illumination control facility, clinically usable chemical derivatives for more specific physicochemical imaging do not yet exist, ICG injection solution contains some sodium iodide; thus, an allergic reaction is possible, ICG is unstable in solutions (10 h) and when exposed to light, and ICG has nonlinear fluorescence quantum yield versus concentration.
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 [
Indocyanine green is a tricarbocyanine dye having a molecular weight of 751.4 Da. It is a negatively charged ion that belongs to the large family of cyanine dyes [
In aqueous solutions, ICG molecules tend to aggregate, which influences their optical properties [
When excitated ICG is supposed to produce singlet oxygen, which is a strongly cytotoxic agent. Engel et al. have recently studied the stability of ICG when exposed to light and the production or the consequences of singlet oxygen production of ICG [
Engel et al. tested several solvents for light-induced decomposition of ICG. What is again interesting and encouraging 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 compared to ICG in water. They suggest that the singlet oxygen produced is quenched by some plasma proteins thus inhibiting ICG decomposition by singlet oxygen [
The important property of fast binding to plasma proteins, especially lipoproteins, [
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 [
The typical dye concentrations used for
ICG cytotoxicity studies.
Cell | Type | Ref. | Comment |
---|---|---|---|
ARPE19 | [ | ICG 1 mg/mL; not toxic | |
RPE | [ | IfCG; no damage | |
Glial cell | Ditto | Ditto | ditto; some damage |
RPE | Rabbit | [ | ICG; no damage |
RPE | Ditto | Ditto | IfCG; no damage |
RPE | Gene expr. | [ | Cell cycle arrest and apoptosis |
Ditto | Ditto | Ditto | 0.25 mg/mL recommended |
Müller | [ | Fluorescent lamp illumination: | |
Ditto | Ditto | Ditto | induces cytotoxicity |
Intravenous | Rat | [ | IRDye 800CW; no toxicity obs. |
Intradermal | Ditto | Ditto | Ditto; ditto |
Spin. root ax. | Rat | [ | Neurotoxicity observed |
RPE | Human cult. | [ | Growth inhibition and damage |
RPE | Human cult. | [ | ICG interactions with RPE |
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 [
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 lipoproteins [
Several encapsulations have been implemented with ICG [
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 [
In this section, an overview of ICG imaging from the instrumentation engineering point of view will be given. Indocyanine green imaging belongs to the class of optical fluorescence imaging. Correspondingly, when used with an operational microscope it closely resembles fluorescence microscopy. Thus, the instrumentation needed is similar or even exactly the same as that for fluorescent imaging in general, or fluorescence microscopy in particular.
As a rule, fluorescence microscopy 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 generally a computationally hard image processing 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 the sensors [
ICGI instruments. KAIST: Korea Advanced Institute of Science and Technology.
Manufacturer | Device | Ref. | Comment |
---|---|---|---|
Carl Zeiss | Pentero IR-800 | [ | Surgical micr. |
Carl Zeiss | Pentero? | [ | Neurosurg. micr. |
Cri, Inc. | Maestro | [ | Small animal |
Eastman Kodak | Imaging Station FX | [ | Small animal |
Eastman Kodak | Ditto 4000 MM | [ | Small animal |
Florida Int. U. | Prototype | [ | Breast imager |
Hamamatsu | PDE | [ | |
Hamamatsu | Prototype ( | [ | Laparoscopic |
KAIST | Prototype | [ | Small animal |
Mizuho Ikakogyo | HyperEye | [ | surgery |
Novadaq Tech. | SPY | [ | |
Osaka Med. Coll. | Prototype | [ | Neurosurg. micr. |
Pulsion | IC-View | [ | |
Topcon | TRC-50IX | [ | Ophthalmoscope |
U. Clinic Munich | Prototype | [ | Endoscope |
U. Kent | Prototype | [ | OCT ophthalm. |
Vieworks Corp. | VasView | [ | Human leg im. |
Wetzlar | Leica OH3 FL800 | [ | Surgical micr. |
ICGI instrument properties. *: Hitachi,
Device | Light | Camera | Ref. | ||
---|---|---|---|---|---|
LED | 740 | cCCD | 820 | [ | |
FX | Halogen | 755 | cCCD | 830 | [ |
Xenon | ? | CCD | 810 | [ | |
IC-View | LED? | 780 | CCD? | 835 | [ |
Maestro | ? | 710 | ? | 800 | [ |
Halogen | 760 | KP-160* | 820 | [ | |
Pentero? | Laser | 780 | ? | 835 | [ |
PDE | LED | 760 | CCD | 820 | [ |
VasView | LED | 760 | CCD | 830 | [ |
SLD | 793 | ? | 807 | [ |
The transmission of the ICG filter pair (
The principle of fluorescence imaging. The radiation from the light source is filtered by a high-pass filter,
In this section, we will, in principle, design a simple ICGA device. The principle of fluorescence imaging is given in Figure
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, MT9V032 is a CMOS sensor for surveillance, KAI-11002 is a standard CCD sensor, and MT9P031 is a standard consumer CMOS sensor.
Table
Light source properties.
Property | Halogen | LED | Diode laser |
---|---|---|---|
Wavelengths | Visual-NIR | Rather narrow | Monochromatic |
Price | Cheap | Cheap | Relatively expensive |
Maintenance | Some | Not much | Some |
Power | High | Rather high | High (pulses) |
Pulses | Mechanically | Electronically | Electronically |
Speed | Slow | Quite fast | Slow-very fast |
Stability | Poor | Good | Good-very good |
Special | Visual imaging | Small size | Extreme performance |
Benefits | Cheap | Easy to control | No filtering needed |
Drawbacks | High power loss | Filter needed | Speckle pattern |
Filter needed | New tech. | White light needed |
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.
Figure
Every CCD or CMOS camera is, in principle, able to record NIR. However, most cameras are prevented from doing so by a filter that cuts NIR wavelengths, otherwise 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 quantum efficiency. The parameters affecting the SNR are the 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
Some commercial NIR camera sensors.
Sensor | Technology | Resolution | Application | Manufacturer |
---|---|---|---|---|
MT9P031 | CMOS | 5 Mpix | Consumer | Aptina |
MT9V032 | CMOS | 0.36 Mpix | Surveillance | Aptina |
KAI-11002 | CCD | 10 Mpix | Consumer | Kodak |
Neo | sCMOS | 5.5 Mpix | Scientific | Andor |
iXon3 | EM-CCD | 1 Mpix | Scientific | Andor |
FL-280 | sCMOS | 2.8 Mpix | Medical | Hamamatsu |
ER-150 | CCD | 1.3 Mpix | Medical | Hamamatsu |
The most important pixel parameters of the above NIR camera sensors.
Sensor | ADC | Read | Dark | Pixel | Well |
---|---|---|---|---|---|
resolution | noise | current | size | depth | |
(bit) | ( | ( | |||
MT9P031 | 12 | 2.6 | 25 | 4.8 | 8.5 |
MT9V032 | 10 | — | — | 36 | — |
KAI-11002 | 16 | 17 | — | 9 | 60 |
Neo | 16 | 1 | 10 | 6.5 | 25 |
iXon3 | — | <1 | — | 13 | 80 |
FL280 | 12 | 3 | — | 13 | 18 |
ER-150 | 12 | 10 | — | 41 | 15 |
An example of the light attenuation in an ICG imaging system.
Row | Component | Attenuation | Remaining intensity | |
---|---|---|---|---|
1 | LED 780-66-60 | 0.2 | ||
2 | Fs, ET775_50x | 0.8 | ||
3 | Tissue irradiance | |||
4 | Tissue, 1 mm | 0.45 | ||
5 | Fluorescence | 0.0027 | ||
6 | Tissue, 1 mm | 0.45 | | |
7 | Lambertian S. | 0.32 | ||
8 | Fc, ET845_55 m | 0.3 | ||
9 | Irradiance, f/1.1 | 0.82 | ||
Response of the Hamamatsu ER-150 low light | ||||
10 | Power per pixel | |||
11 | Photons per pixel | |||
12 | Detected photons | 0.25 | ||
13 | Optimal exposure | |||
14 | Signal to noise | SNR = 63 dB |
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 efficiency tends to decrease quite rapidly by increasing wavelength (Figure
For the LED source of Figure
A rigorous approach to ICGA system design would include 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 enough.
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
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 [
The fluorescent light proceeds through the layer of tissue, which again attenuates the irradiance by the factor of
Before hitting the sensor, the radiant intensity,
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,
The optimal exposure time
As an example, the calculation of the observed fluorescent intensity and the performance of the Hamamatsu ER-150 sensor is estimated in Table
The loss factors and corresponding attenuations [dB] of the top five loss factors in ICGA imaging.
Loss factor | Loss | dB |
---|---|---|
The fluorescence of ICG in blood | 0.0027 | −25.6 |
Losses in the tissue above the blood vessel | 0.2 | −6.9 |
Quantum efficiency of sensor | 0.25 | −6.0 |
Transmittance of the emission filter | 0.3 | −5.2 |
Diffusion losses in the lambertian surface | 0.32 | −5.0 |
Subtotal | −48.7 | |
Other factors together | 0.62 | −1.7 |
Total | −50.5 |
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 880 nm, as a test light to see if the camera is tuned to wavelengths ranging from about 800 nm to 900 nm. 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 fluorescing themselves.
Our example system was based on an old operational microscope originally not at all designed for NIR imaging (Wild, Figure
An old operational microscope used in our prototype ICG stereo video angiography system experiment. Hamamatsu NIR camera on the left camera arm.
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
After technical laboratory tests, our device was tested by recording ICGA of rat heart (Figure
Using two cameras, stereo images and stereo video can be taken. The cameras can be attached to an operational microscope (Figure
Established medical applications of ICG are retinal angiography, 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 [
As compared to other angiography 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 minutes after injection. Normally a new bolus can be given after about 15 minutes.
Neurosurgery is ideal for ICGA because operations are already done under a microscope (and camera), and because the blood veins located on the brain surface are mainly exposed and thus can be seen more or less directly by visual means. Milestones in neurosurgery include 2001: experiment with surgical microscope (OPMI) in neurosurgery [ 2002: FDA approval for cerebral angiography research [ 2003: ICGA was introduced for clinical neurosurgery [ 2005: ICGA done with surgical microscope [ Leica 2006: FDA approval of ICGA surgical microscope; Zeiss 2007: commercial surgical microscope with ICGA; 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 surgically are cerebral aneurysms and intracranial or intraspinal arteriovenous malformations (AVMs) and fistulas. It is of utmost importance to be able to verify that the malformation 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 possible 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 (<1 mm) perforating arteries, which, despite their small caliber, may supply blood flow to critical neural structures in, for example, basal ganglia 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 [
During microneurosurgical treatment of brain or spinal arteriovenous malformations and dural arteriovenous fistulas, 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 [
ICG angiography has also been evaluated and found reliable in assessing the patency of microanastomoses in neurosurgical extracranial-intracranial revascularisation bypass operations [
The usefulness of ICG angiography in microneurosurgical vascular operations is increasingly acknowledged, as more 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 analyses and easily processable and repeatable video playback loops, since rapid ICG reinjections generally suffer from lower contrast due to residual ICG inside the vessels.
In principle, coronary arteries are also 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 2002: a pig model of coronary bypass angiography with ICG [ 2004-2005: comparison of ICGA and ultrasound flow metering at University of Toronto; 2005: FDA approval for ICGA device SPY for coronary angiography; 2005–2009: GRIIP clinical trial (phase III) at Sunnybrook Health Sciences Centre.
Coronary artery bypass grafting (CABG) is the most frequent cardiac operation with annual rates of 400,000 procedures 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) [
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.
Firstly, an indirect method in which the myocardial tissue 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 [
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 [
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 published 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 [
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 predicting wound healing after major amputation and to evaluate splanchnic circulation. Also, the usefulness of ICG angiography 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 [
In a recent study, Kang et al. tested the use of combined analysis of multiple parameters, especially onset time and modified
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 vessel occlusion in atherosclerosis. It has been recognised that 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 specimens it has been demonstrated that ICG colocalised with lipid-rich atheroma and macrophages. Together these results suggest that ICG may be useful as a imaging agent specifically 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) transcutaneously. Each patient was connected to an ICG finger clip, which was connected to a liver function monitor (LiMon). A 0.25 mg/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. Levosimendan did not have a significant effect on total splanchnic perfusion in patients undergoing an elective aortic aneurysm operation [
Foam sclerotherapy is a widely used treatment for varicose veins. The spreading of the sclerosant is usually visualized 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 [
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 [
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 [
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 completeness of surgical resection and perioperative evaluation of potential dissemination of a malignant and deadly disease.
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 lymphatic pumping with a commercially available device and a custom-made transparent sphygmomanometer [
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 [
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 intraoperationally during a laparoscopic hepatectomy [
In 2009, two clinical studies revealed that real-time ICG-fluorescent imaging enabled the highly sensitive identification of small, grossly unidentifiable liver cancers. This led to an enhanced accuracy of operative staging and liver resection. Currently, indocyanine green fluorescence imaging navigation is considered to be a promising tool for clinical exploration for hepatocellular carcinoma and for routine intraoperative imaging during hepatic resection [
Sentinel lymph node detection has been one main application of ICG in laparoscopic studies including early gastric cancer treatment and gastrectomy [
Note, that CO2 pneumoperitoneum used in laparoscopic operations, which decreases liver blood flow, also increases ICG half-life [
While ICG is rapidly excreted via the bile duct, it is most natural to apply ICG intraoperationally to aid bile examination and operations [
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 × 30 cm 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 perfusion 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 [
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
In addition to the above surgical applications, the clinical application of ICG include such topics as brain imaging and hemodynamics [
Due to the absorption at wavelengths under 800 nm, ICG can be used with a suitable intense light source, typically a laser, as an ingredient of a tissue solder like albumin, which readily bounds to ICG [
When an ICG molecule is excited, it can further transfer energy to other molecules. When exciting oxygen, ICG turns out to be a photodynamic therapy agent. In principle, for example, after having been used to reveal lymph nodes a strong illumination 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
Similarly, ICGA can be used as a light-activated antibacterial agent (LAAA), for example, in wound healing [
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 indocyanine green is one of the prospective exogenous dyes for soft photodynamic treatment (PDT).
Finally, as ICG is a dye it can be naturally used for tattooing, labeling, and similar tasks [
Because hepatocytes handle ICG in the liver, ICG can be used also to monitor differentiation of mouse embryonic stem cells into hepatocytes [
In this work, we have reviewed well over 200 papers describing the development and use of the fluorescing contrast agent 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 succeeded in collecting most of the key publications for giving an overview of the indocyanine green fluorescence technology and its most important emerging clinical application.
Many new clinical applications of ICG and ICG angiography are just emerging and more are definitely expected to appear in the near future; thus, it is obvious that much more research is still needed in order to fully realise all the potential of this relatively simple optical technology. The obvious fields of further engineering research include image processing of ICG and ICGA information, also in real-time (video and stereo) [ capillary circulation monitoring and perfusion dynamics imaging [ combining ICG and other imaging modalities like visual, CT, MRI, and PET [ combining ICG and ICGA with dermal imaging methods [ deeper imaging (optical tomography) [ optical imaging device development (laparoscopy) and optimisation, hands-free [ development of new derivatives of ICG for more specific imaging modes, increasing the quantum efficiency of ICG by, for example, metallic nanoparticles [ micro- and nano-encapsulation of ICG for nonangiography applications [ extraction of spectral information and chemometry (multispectral imaging) [ integration of ICG imaging to robotic-assisted surgery [
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 applications [
Three-dimensional
Anterolateral thigh flap
American Society of Anesthesiology Patient Classification Status
Arteriovenous malformation
Blood pressure
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
High-density lipoprotein
Human serum albumin
Helsinki University Central Hospital
Independent component analysis
Image-intensified CCD
Indocyanine green
ICG plasma disappearance rate
ICG video angiography
ICG angiography
ICG imaging
ICG plasma disappearance rate
Intensive care unit
Infracyanine green
Infrared-ray electronic endoscopy
In situ photoImmunotherapy
Light-activated antimicrobial agents
Low-density lipoprotein
Laser Doppler perfusion imaging
Light-emitting diode
Laparoscopic intragastric full-thickness excision
Molecular Imaging and Contrast Agent Database
Magnetic resonance imaging
Near-infrared laser illumination
Near-infra red
NIR fluorescence
NIR spectroscopy
Natural orifice translumenal endoscopic surgery
Optical coherence tomography
Peripheral arterial disease
Peripheral arterial occlusive disease
Sodium polyaspartate
Phosphate-buffered saline
Photodynamic therapy
Photoplethysmography
Photothermal therapy
Peripheral vasculature
PV disease
Quantum dot
Red blood cell
Red green blue
Raynaud phenomenon
Retinal pigment epithelium
Superluminescent diode
Sentinel lymph node
Sentinel lymph node biopsy
Sentinel node navigation surgery
Signal-to-noise ratio
Transit-time flowmetry.
The FIELD NIRce project and especially Professor Paul Geladi at SLU University are acknowledged for both their economic and scientific support (J. T. Alander and P. Välisuo) 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) [