Abstract

Inside the brain tumor, the blood vessels are intricately composed, and the tumors and blood vessels are similar in color. Therefore, when observing tumors and blood vessels with the naked eye or a surgical microscope, it is difficult to distinguish between tumors and blood vessels. Fluorescence staining with indocyanine green (ICG) is performed to distinguish between brain tumors and blood vessels using a surgical microscope. However, when observing the blood circulation state of a tumor or blood vessel through a surgical microscope, light reflection occurs from the camera. In the process of observing the state of the blood vessel, due to the occurrence of light reflection, an obstruction phenomenon in which the observation field is blocked by the blood vessel of the object to be observed occurs. Therefore, it is difficult to diagnose the vascular condition. In this experiment, the 780 nm light-emitting diode (LED) was irradiated to the ICG phantom, and then, when the fluorescence expression image was observed, the polarizing filter such as circular polarized light (CPL) filter and linear polarized light (LPL) filter was inserted into the camera, and the reflected light was reduced. Therefore, it is possible to reduce the reflected light from the fluorescence expression image by using a polarizing filter, and it is expected to be applicable to surgery and diagnostic fields of cancer such as surgery.

Introduction

Cancer is the leading cause of death worldwide, with 18.1 × 106 cancer patients in 2018. In Korea, the number of cancer patients in 2017 was 232,255, an increase of 1019 compared to the previous year [1].

Malignant tumors have strong invasiveness. Accordingly, complete resection of the tumor is of great importance in cancer surgery. However, a tumor has a large amount of blood vessels, and the color of the blood vessels is similar to that of the tumor, so it is difficult to distinguish the boundary between the tumor and blood vessels through visual observation or surgical microscope observation [2].

Thus, in cancer surgery, a fluorescent staining diagnostic method is used to clearly distinguish the boundary between a tumor and a blood vessel. The fluorescent staining diagnosis method is a method in which fluorescence in tumors or blood vessels is induced by orally or intravenously injected with a fluorescent contrast agent, making the boundaries of tumors and vessels clearly observable by external monitors [3]. The device used for the fluorescent staining diagnostic method is a surgical microscope or a camera, and the fluorescent contrast agent is divided into fluorescein sodium, 5-aminolevulinic acid, and indocyanine green (ICG). Among them, a fluorescent contrast agent for observing blood circulation of blood vessels is ICG.

However, when light of a specific wavelength is irradiated, polarized reflected light is generated in the blood vessel due to the difference in refractive index of the medium. Due to the reflected light, it is difficult to secure a view of the blood vessels during surgery, making it difficult to diagnose the blood circulation status of blood vessels [4].

In this experiment, a polarizing filter was used as a principle of reducing light in a specific direction from the viewpoint of polarization of reflected light. In this paper, we propose a fluorescence staining diagnostic method using a polarizing filter to increase the accuracy of tissue diagnosis by reducing the reflected light to secure a field of view to observe the blood flow state of blood vessels during surgery.

Theory of Polarization and Experiment Configuration

Analysis of Polarizing Filter.

A polarizing filter is a filter that reduces linearly polarized reflected light on the surface by passing only light that vibrates in a specific direction [5]. Polarizing filters are classified into linear polarized (LPL) filters and circular polarized light (CPL) filters. The LPL filter is a linear polarization filter that transmits light in one direction as shown in Fig. 1 [6,7].

Fig. 1
Actuation principle and structure of polarized light filter
Fig. 1
Actuation principle and structure of polarized light filter

As shown in Fig. 2, the CPL filter generates circulation polarization when incident light is linear polarization, and if CPL light is incident on the CPL filter, the light does not pass [8]. As shown in Fig. 3, the light, which is linearly polarized at 45 deg because of the polarized sheet, is polarized by two refraction waves from the interface of the phase retardation plate. When the ratio of the refractive index to the polarized wave of the x-axis and the y-axis is 3: 4, and the ratio of the thickness and wavelength of the uniaxial crystal is fixed to 1: 2, a phase difference of π/2 (90 deg) occurs [9,10].

Fig. 2
Actuation principle and structure of circular polarized light filter
Fig. 2
Actuation principle and structure of circular polarized light filter
Fig. 3
Block diagram of polarizing process
Fig. 3
Block diagram of polarizing process
In Eq. (1), εx and εy are the wavelengths for the x-axis and y-axis, respectively, ax and ay are the magnitudes of the wavelengths, and Δ is the phase difference. Analyzing Eq. (1), the shape of polarization changes according to the phase difference, and the result can be confirmed through Fig. 4. In addition, when the simulation is performed through the above principle, the results shown in Fig. 5 can be obtained.
εx2+εy2acos(Δ)εxεy=axaysin2(Δ)
(1)
Fig. 4
Polarization pattern depending on phase difference
Fig. 4
Polarization pattern depending on phase difference
Fig. 5
Propagation of circular polarization (a) cross section of optical axis and (b) combined fields
Fig. 5
Propagation of circular polarization (a) cross section of optical axis and (b) combined fields

Experiment System.

This experiment is intended to reduce reflected light when ICG fluorescence is expressed using a polarizing filter. For fluorescence expression and analysis, a 780 nm light-emitting diode (LED) (Thorlabs, M780L3 and Bandwidth (FWHM) of 28 nm) and a near infrared camera were used. Table 1 shows the specifications of the camera [11]. In order to improve the image quality of fluorescence-expressing images, high-pass filters (Thorlabs (Newton, NJ), FEL0800) with reflectance and transmittance of 0.01% and 98%, respectively, were inserted into the camera [12]. Therefore, the wavelength of 800 nm, which is infrared, is passed through the camera. In addition, to reduce reflected light, a LPL filter (Kenko) and a CPL filter (MountRation) were connected to a camera lens. The LED used as the irradiation light source has a wide beam width as shown in Fig. 6, and the beam width value is 20 deg [13].

Fig. 6
Beam pattern for radiation of 780 nm LED
Fig. 6
Beam pattern for radiation of 780 nm LED
Table 1

Specifications of near-infrared (NIR) cameras

TypeModel nameSpecification
NIRLt-225M-NIR-SCPass range (nm)400–1000
Pixel size (μm)5.5 × 5.5
Resolution (pixels)2048 × 1088
Frame rate (fps)170
TypeModel nameSpecification
NIRLt-225M-NIR-SCPass range (nm)400–1000
Pixel size (μm)5.5 × 5.5
Resolution (pixels)2048 × 1088
Frame rate (fps)170

The distance between the phantom of the fluorescent contrast agent and the light source was fixed to 5 cm. As shown in Fig. 7, when fluorescence is observed, the LED and the phantom are fixed vertically, and the camera is maintained at 72 deg to the phantom.

Fig. 7
Configuration of experimental system (a) angle of irradiation source and camera and (b) image of experimental conditions
Fig. 7
Configuration of experimental system (a) angle of irradiation source and camera and (b) image of experimental conditions

To manufacture a phantom capable of fluorescence expression, ICG 25 mg (molecular weight: 774.96 g/mol) is injected with 10 ml of normal saline to prepare the reagent at a concentration of 2.5 mg/ml, that is, 3.23 mM. At this time, in order to fix the ICG concentration at 10 μM, 0.155 ml of the prepared ICG reagent was injected into 50 ml of Latex rubber [14].

Experimental Results.

When light is irradiated at a material, incident light, reflected light, and refracted light are generated according to Snell's law of refraction. In Fig. 8, assuming that the refractive indices of the two media are n1 and n2, respectively, Eq. (2) is satisfied [7]. Then, polarization occurs from the surface. As shown in Fig. 8, when the angle between the refracted and reflected light is 90 deg, the reflected light is polarized according to Brewster's law
n1sinθ1=n2sinθ2
(2)
Fig. 8
Light incident on the interface of two media
Fig. 8
Light incident on the interface of two media

In this experiment, fluorescence expression was observed for ICG through irradiation of 780 nm LED as shown in Fig. 7(a). The power of the irradiation light source measured by the power meter is 200 mW, and the peak emission wavelength of the LED and the peak emission wavelength for the ICG measured through the spectrometer are 780 nm and 810 nm, respectively.

When observing fluorescence after investigating specific wavelengths for ICG phantom, fluorescence expression images could be analyzed as shown in Figs. 911, respectively, depending on the presence or absence of a polarization filter. In Figs. 10 and 11(a), it was confirmed that the area of the reflected light indicated by the dotted line in Fig. 9 decreases when the CPL filter and the LPL filter are used. Finally, Fig. 11 shows the oral (tongue) image for the practical performance test of the filter. The subject was the author's tissue, and light reflection occurred when there was no filter, but it was confirmed that light reflection was reduced by 99% when the filter was present.

Fig. 9
Experimental results for ICG phantom without polarizing filter (a) original image and (b) mark for area of reflected light
Fig. 9
Experimental results for ICG phantom without polarizing filter (a) original image and (b) mark for area of reflected light
Fig. 10
Comparison of fluorescence expression images according to polarizing filters (a) without polarizing filter and (b) with CPL filter
Fig. 10
Comparison of fluorescence expression images according to polarizing filters (a) without polarizing filter and (b) with CPL filter
Fig. 11
Comparison of fluorescence expression images according to polarizing filters (a) without polarizing filter, (b) with LPL filter, and (c) human tissue test (tongue)
Fig. 11
Comparison of fluorescence expression images according to polarizing filters (a) without polarizing filter, (b) with LPL filter, and (c) human tissue test (tongue)

Discussion.

The fluorescence-expressing images are obtained through a surgical microscope during surgery using a fluorescent staining diagnostic method. Ideally, if only the emission wavelength is incident on the microscope lens, it will help to accurately diagnose the blood flow condition with clear image quality. However, when fluorescence-expressing images are observed, ambient external light sources are incident on the lens together with the emission wavelength. Therefore, as shown in Fig. 12, a high-pass filter (Thorlabs, FEL0800) was inserted into the head portion of the camera to improve the image quality of the diagnostic image by reducing the surrounding interference wavelength.

Fig. 12
Internal composition of a camera
Fig. 12
Internal composition of a camera

In addition, the fluorescence contrast agent used in this experiment is indocyanine green (drug name: DID-Indocyanine Green inj), and prior to the experiment with fluorescence expression in animal and patient tissues, the experiment on fluorescence expression in phantom with fluorescence contrast agent was conducted to report the possibility of the proposed method. Since positive results were obtained through this experiment, the need for animal and clinical trials emerges based on the proposed method.

The results of the above experiment show that when polarizing filters are used, the reflective light is reduced in fluorescent expression. Also, it can be seen that when the LPL filter is inserted, the reduction of reflected light is greater than when the CPL filter is inserted. This is because the camera used in the experiment was not an autofocus (AF) model. The CPL filter was developed because of the AF technology of a single lens reflex (SLR) camera. Light entering through the lens of the SLR camera is reflected to the viewfinder because of the internal mirror placed. At this time, all the light entering the lens is not reflected, and some of them move to the AF sensor in the camera to focus [15,16]. That is, a phase retardation plate is included to prevent double polarization because it adjusts the focus using polarized light. Thus, in the case of an non-AF type camera, when using a CPL filter, the effect of reducing reflected light is less than that of a LPL filter. In other words, it is necessary to distinguish the use of filters according to the built-in focus method of the camera. For example, the camera may use a manual focus control function, but the filter is considered to have to use CPL and LPL together.

Conclusion

Fluorescent staining using an ICG contrast agent is used to accurately diagnose the blood flow status of blood vessels during cancer surgery for complete resection. However, there is a problem in that it is difficult to secure a surgical field because of reflection of light at the interface.

In this paper, ICG fluorescence contrast medium injection is inserted into a surgical diagnostic camera in the surgical procedure for tumor removal to reduce light reflection and thus vascular status (vascular damage or blood circulation status during surgery). We suggest a method to secure the field of view for diagnosis. In this experiment, the polarizing filters were used to confirm the reduction of reflected light in the fluorescence expression image for ICG, thereby proving its potential.

In conclusion, the diagnostic method for fluorescence staining of ICG using a polarizing filter proposed in this paper will be helpful in accurately diagnosing the condition of blood vessels and blood circulation during tumor removal surgery. It will also help the operator to secure a view of the blood vessels. In addition, it is expected that the operator will help to proceed with the operation quickly and accurately. Furthermore, it is considered to be of great help to the treatment of cancer in department of radiation oncology and surgery in the future.

Funding Data

  • Research and Development for Regional Industry Seoul R&D Program (Grant No. BT190153).

  • Ministry of SMEs and Startups Technology Development Program (MSS, Korea) (Grant No. S2797147).

Nomenclature

     
  • ax =

    x-axis magnitude

  •  
  • ay =

    y-axis magnitude

  •  
  • AF =

    auto focus

  •  
  • SLR =

    single lens reflex

  •  
  • εx =

    x-axis wavelength

  •  
  • εy =

    y-axis wavelength

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