Glioma, one of the most common primary brain tumors, shows malignant progression characterized by early widespread invasion throughout the brain [1].

To date, the best treatment for malignant gliomas is extensive tumor resection, when possible, accompanied by radiotherapy and chemotherapy [2, 3]. The two most common systems used for brain tumor observation are MRI and surgical microscopes. However, it is not easy to carry out complete resection of glioma because the interface between the tumor and normal brain tissue is difficult to identify with a conventional surgical microscope [4], due to its poor picture quality. Inaccurate information about the location of a tumor may lead to excessive normal tissue sacrifice or even cancer recurrence [5-9]. Therefore, in order to overcome these problems, it is necessary to develop a fluorescence guided operation system consisting of an LED which can detect fluorescence in blood by irradiating cancer with the LED to easily identify blood vessels and tumors through fluorescent color. Recently, several groups have studied fluorescence imaging systems and fluorescence biomarkers for clinical applications and many preclinical and clinical studies have shown the possibility of fluorescenceguided surgery in malignant gliomas. Some fluorescent dyes used in brain tumor surgery are 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) [10-12].

5-ALA is the most studied and the most used of the various fluorescent tumor markers for fluorescence-guided surgery. When 5-ALA fluorescent dye is taken orally, it is converted into protoporphyrin IX (PpIX) by a metabolism. Malignant gliomas produce and accumulate excessive amounts of intracellular PpIX compared to the surrounding normal brain parenchyma. The PpIX within the tumor emits red fluorescence under a violet-blue light. Thus, red fluorescence highlighting viable tumor cells is visualized on a dark blue-green background. Furthermore, clinical trials have shown that this surgical method is more convenient for tumor resection and enables better resection of the tumor boundary, which improves the survival rate. However, one of the major problems during 5-ALA fluorescence-guided surgery is the dark surgical field [11, 12], leading to some possible major vascular injury [13, 14]. On the other hand, Indocyanine Green (ICG) is a fluorescent material which is effective to determine the position of the patient’s blood vessels with few side effects. Upon injection, ICG combines with plasma proteins [1].

The developed multi-fluorescein emission system is composed of LEDs, a beam prism, dichroic mirror, emission filter, excitation filter, mirror, and CCD camera as shown in Fig. 1. From the figure, the LEDs used are FD-3535IR770 and Epileds 5050 10 W UV LED Emitter Lamp Light which have wavelengths of 740 nm and 410 nm, respectively. In addition, the fluorescein microscopy uses a Perkin Elmer Solaris, combined with the beam prismbeam splitter module which mounted the filter to the microscope (zoom of 12.5 @ ophthalmic surgery microscope/on casters) in response to the fluorescent dye. A fluorescence image was confirmed through the display device with combined video adapter, which mounted a CCD camera (SBIG STF-8300C Color CCD Camera).

The system specifications of Fig. 1, the 740 nm and 410 nm of the LEDs are shown in Table 1. Also, the specification of the CCD camera (SBIG STF-8300C Color CCD Camera in OPT the telescope authority) is shown in Table 2. The filter is used for filter of fluorescein wavelengths at 410 nm and 740 nm and the filtering of fluorescein has increased resolution. Also, the beam splitter is a 10BC68-1-UV which has wavelength range of 19 nm to 2200 nm. 5-ALA itself is not a photosensitizer, but it is a precursor of a strong photosensitizer, protoporphyrin IX (PpIX), which reacts to the tumor cells by emitting a strong fluorescence. In contrast, ICG has a self-light emitting property.

Figure 2(a) shows the experimental results for excitation wavelength of the 410 nm and 740 nm at LEDs which is used for irradiation wavelength for fluorescein emission wavelength of 5-ALA and ICG. The light source was attached to an existing surgical microscope front end with a ring shape as shown in Fig. 2(b), which makes possible to emit the light. The light source was constructed with an 8 cell LED with 410 nm and 740 nm wavelengths of light. The LEDs have wavelengths of 410 nm and 740 nm, respectively. Fluorescent images were obtained using a beam splitter and a near-infrared camera. Filters are configured with an optical apparatus that was optimized for each of the wavelengths in order to determine the fluorescence image acquired from the microscope using a CCD camera. The right side of the module has the emission wavelength of the 5-ALA 628 nm and the left side is a fluorescent wavelength of ICG 825 nm, in order to show the best efficiency configured with the optical device and the filter. Optics and light sources used in the filter module are shown in Table 3. Filters used for the wavelength emission were 410~760 nm filter and 628~825 nm filter for 5-ALA and ICG respectively.

In addition, the filter module has an advantage that it can be attached to the existing microscope. A filter module was configured with a transmissivity between 430~670 nm to transmit the emission wavelength of the 5-ALA and the ICG, which has more than 90% of transmittance rate in between 720~1080 nm of wavelength range. In the configuration of the filter, both transmittances of 90% were used. (ICG: 825 nm, 5-ALA: 628 nm).

Figure 3 shows an LED module (410 nm) attached to a surgical microscope to observe (628 nm, bandwidth: 32 nm) an irradiated target with a light source. Figure 4 is the photograph of the observation described in Fig. 3. The animal experiments were conducted in compliance with the deliberations provisions of the animal experimentation ethics committee of the National Cancer Center (NCC-14-219). In preparation for an experiment, a nude mouse (26 g) was housed three to four weeks for tumor growth. Then we injected 5-ALA of 7.5 mg/150 μl intravenously 6 hours prior to an experiment. Results were obtained through a video format recording.

Figure 5 is the graph which shows the measurement result of excitation wavelength band and emission wavelength band when an LED light source (ICG: 740 nm, 5-ALA (Protoporphyrin IX): 410 nm) was emitted. The one peak point was seen in the emission wavelength range with an ICG solution and some part of the range was overlapped with the range of excitation wavelength.

The LED is considered a significant factor for selection of the filter because its wavelength is not in the short wavelength region but it is in the region of gentle curves. There were 2 peak points in the emission wavelength range of 5-ALA (Protoporphyrin IX) solution, unlike ICG solution. And the range covers a relatively wide region about 100 nm. As its excitation wavelength band is 200 nm away from the range of its emission wavelength, 5-ALA is more flexible to be selected as a filter than ICG.

The ICG is emitted the fluorescein at above 800 nm It has tricarbocyanin dye with peak spectral absorption. Following intravenous injection, the ICG is rapidly bound to plasma protein of which albumin is the principle carrier by 95%. The ICG is taken up from the plasma almost exclusively by the liver and is secreted entirely into the bile as shown in Fig. 6 [15]. 5-ALA is changed to PpIX in tumor via an oral-intake of exogenous 5-ALA. In the 410 nm optical source with the specific wavelength, PpIX fluorescence acts as shown in Fig. 7 [16].

For animal experiments, the possibility of fluorescence expression of brain tumors was examined in mice. Figure 8 shows experimental results of fluorescence wavelength of 825 nm and red fluorescence color in blood vessels when a 740 nm LED beam was irradiated on tumor and blood vessels by contrasting the ICG fluorescence. Figure 8 shows the fluorescence emission from the tumor when irradiated with 410 nm LED beam to tumors and blood vessels by injecting 5-ALA fluorescent substance into the tumor. The fluorescence intensity of 628 nm and purple fluorescence color was displayed. From the figure, the tissue is separated between blood vessel and tumors. The tumors is clearly expressed through the 5-ALA fluorescein dye. The white color area is fluorescein emission of PpIX ICG substance in the blood of the mouse. Then, the nude mouse (26 g) was housed four weeks for tumor growth. Also, we injected 5-ALA of 7.5 mg/150 μl intravenously 6 hours prior to an experiment. The ICG was injected concentration of 0.5 mg/kg and injection volume of 0.5 ml. An ICG was injected into the tail vein of the mouse after observing the fluorescence with a microscope. The weight of the mouse is 26 g. Results were obtained through a video format recording.

Therefore, the newly developed multi-detection microscope allows accurate visual observation of tumor and blood vessel morphology through fluorescence color, so that the margin of the tumor can be confirmed.

A real-time distinction between glioma and normal brain tissue will be of great help to neurosurgeons. Recently, the fluorescence guided brain tumor surgery has evolved into a powerful means of distinguishing gliomas from normal brain tissues intraoperatively. Currently, there are two types of fluorescence imaging systems used in clinical practice: 5-ALA and ICG. 5-ALA has been used to detect tumors and ICG mainly has been used to observe blood vessels and has recently been shown to be helpful in tumor detection.

A near-infrared (NIR) fluorescent dye, 5-ALA and ICG has an excitation wavelength of 410 nm, 740 nm, and emits 628 nm, 825 nm wavelength of light, respectively. These dyes have been widely used in vascular surgeries. This is the fluorescent material which is effective to determine the position of the patient’s blood vessels. ICG angiography especially can provide real-time information during the surgical management of intracranial aneurysms, arteriovenous malformation, and bypass procedure.

According to the result of a randomized controlled multicenter phase III trial, 5-ALA fluorescence-guided resection increased gross total resection rate and progression-free survival compared to standard microsurgery. However, 5-ALA fluorescence-guided surgery has some disadvantages. One of the major problems in 5-ALA fluorescence-guided surgery is the low intensity of blue light for background illumination. Weak illumination increases the risk of resection of adjacent brain tissue and damage of major blood vessels. In order to maximize safe resection of brain tumors without complications, tumor mass and adjacent major blood vessels should be identified by the surgeons simultaneously.

To detect the fluorescent images of 5-ALA and ICG, we have developed a dual light source using a 410 nm and 740 nm LED and a CCD video camera with appropriate filters which can be attached to a conventional neurosurgical operating microscope. Using this apparatus, 5-ALA and ICG fluorescence can be observed separately or simultaneously. Moreover, we have produced a separate beam splitter module which could be mounted on the surgical microscope in order to supplement lacking path of light. The filtered images can be transmitted to a camera with the filter for respective emission wavelength. The wavelength of the filter was fixed as 628 nm and 825 nm. And we carried out animal experiments using rats and nude mice for the performance test of the proposed system. We validated the observability of tiny tumor and small size blood vessels by using animals smaller than humans for the experiments.

It was verified that the images obtained on the left row were not exactly matched with the ones on the right row due to the nature of the surgical microscope.

We confirmed various parameters while we were developing the LED module. When LED power is not enough, we were not able to obtain the fluorescence image. And in the opposite condition, it was impossible to observe the fluorescence image because of the wavelength shift phenomenon caused by high power. When the temperature of the LED semiconductor chip exceeds 80°C, the limit of its operation temperature, a wavelength shift phenomenon occurs. As a solution, we have configured the cooling system using a radiator and a cooling fan.

There are frame rate and exposure time as variables on the camera setting value. High fluorescence image can be acquired when the exposure time is set to the maximum value. Mostly quantum efficiency value graph is considered as main criteria for selection of a camera. But it can’t be the absolute criteria, as quantum efficiency value is measured by different ways according to camera companies. Therefore, we chose the camera which showed the highest fluorescent sensitivity at the wavelength we desired through numerous camera tests.

The lifetime of ICG continues for only 5 minutes while the lifetime of 5-ALA lasts for 24 hours after administration. In addition, the fluorescence emission of ICG is observable right after administration while fluorescence emission of 5-ALA is detected 2~6 hours later. We have solved these differences through the image-processing algorithm and made it possible to observe both of them simultaneously.

In a sample test, The fluorescence emission of the reagents was detected immediately in a sample test which was made suitable for observation. Due to variable in animal experiments, it took a long time.

Thus, in this study, we developed the system in order to detect blood vessels and tubmors in both florescence of 5-ALA and ICG in the surgical microscope and these can be effectively observed.

The tumor resection is in parallel with radiation therapy and chemotherapy in the treatment of glioma. However, the distinction between the normal tissue and tumor is difficult to differentiate with a conventional surgical microscope. Thus, we have developed a new surgical fluorescence microscope to detect both 5-aminolevulinic acid (5-ALA) and indocyanine green (ICG) fluorescence materials. The suggested microscope was coupled to a two types light emitting diode (LED) which has a wavelength of 410 nm and 740 nm. This microscope includes beam splitter and filter for obtaining clear tissue imaging. The lifetime of ICG continues for only 5 minutes while the lifetime of 5-ALA lasts for 24 hours after administration. In addition, the fluorescence emission of ICG is observable right after administration while fluorescence emission of 5-ALA is detected 2~6 hours later. We have solved these differences through the image-processing algorithm and made it possible to observe both of them simultaneously.

The concentration was made suitable for observation when the fluorescence emission of the reagents was detected immediately in a sample. But, due to many parameters in animal experiments, it took a long time to observe fluorescence. Thus we developed the system that can be effectively observed both fluorescence of 5-ALA and ICG in the surgical microscope to detect blood vessels and tumors.