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A novel laparoscopic near-infrared fluorescence spectrum system with indocyanine green fluorescence overcomes limitations of near-infrared fluorescence image-guided surgery

Background: Near-infrared (NIR) fluorescence image-guided surgery (FIGS) introduces a revolutionary new approach to address this basic challenge in minimally invasive surgery. However, current FIGS systems have some limitations – the infrared rays cannot detect and visualise thick tissues with low concentrations of the fluorescent agent. We established a novel laparoscopic fluorescence spectrum (LFS) system using indocyanine green (ICG) fluorescence to overcome these limitations. Materials and Methods: Bovine serum albumin (BSA) was conjugated to ICG, and the mixtures were serially diluted at 5 × 10−8–5 × 10−1 mg/mL. We used the LFS system and a NIR camera system (NLS; SHINKO OPTICAL CO., LTD Tokyo, Japan) to determine the optical dilution for the fluorescence detection. BSA was conjugated to ICG (5.0 × 10−2 mg/mL) and used to coat the clips. We attempted to identify the fluorescence-coated clip from the serosal side of the cadaveric porcine stomach tissues using the LFS system and the NIR camera system. We measured the depth of the cadaveric porcine stomach wall at the thickest part that could be confirmed. Results: We could not visualise fluorescence concentrations <2.5 × 10−3 mg/mL using the NIR camera system. The spectrum was detected at a concentration <2.5 × 10−3 mg/mL. We were able to identify the spectrum of ICG (829 nm) to a 13-mm depth of cadaveric porcine stomach wall by using the LFS system but could not identify the same with the NIR camera system regardless of wall thickness. Conclusions: The novel LFS system with NIR fluorescence imaging in this ex vivo and cadaveric porcine model was confirmed useful at deeper depths and lower concentrations. Based on these findings, we anticipate that the LFS system can be integrated and routinely used in minimally invasive surgery.


INTRODUCTION
Near-infrared (NIR) fluorescence imaging has recently experienced rapid expansion on a global scale. With real-time intra-operative NIR fluorescence imaging of tumours and sentinel lymph nodes (SLN), the technique promises to guide the oncologic surgeon towards optimal radical resection and clinical results. [1] NIR fluorescence image-guided surgery (FIGS) introduces a revolutionary new approach to address this basic challenge in minimally invasive surgery. The feasibility of laparoscopic NIR fluorescence imaging systems using indocyanine green (ICG) has recently been reported for the purpose of SLN mapping in various solid cancers. [2][3][4] However, currently these systems have some limitations, including that the infrared rays cannot detect tumours within thick tissue using low concentrations of the fluorescent agent. We established a novel laparoscopic fluorescence imaging and spectrum (LFS) system to overcome these limitations.

Novel laparoscopic near-infrared fluorescence spectrum system
The laser diode (wavelength, 785 nm; maximum output power, 5 mW) was used as the exciting light source. The laser beam for irradiation was focused on the central part of the fused silica coaxial fibre, which has a Y-type configuration [ Figure 1]. A notch filter was used to filter out the reflected laser signal exiting the collecting fibre; thereafter, only the fluorescence signal was detected using the photonic multichannel analyser. The NIR fluorescence excited from the ICG sample was collected through the outer part of the coaxial fibre and measured by spectroscopy. Because the centre wavelength of fluorescence occurs at about 840 nm, which is close to the exciting wavelength of 785 nm, a narrow-band notch filter was used at the front of the input slit of the spectroscope to cut off the signal from the exciting light source. For details about the novel system, see our previous report [5] [ Figure 1].

Near-infrared camera system
We used a NIR camera system (NLS; SHINKO OPTICAL CO., LTD Tokyo, Japan) that activates ICG with emitted light (wavelength, 760 nm).

Fluorescent reagent
ICG, which was purchased from Diichi-Sankyo (Tokyo, Japan), is a popular diagnostic regent approved clinically for the examination of hepatic and circulatory function. [6,7] ICG is a sterile anionic water-soluble but relatively hydrophobic tricarbocyanine molecule with a molecular mass of 776 Daltons. The ICG dye was developed for NIR photography by the Kodak research laboratories in 1955 and approved for clinical use in 1956 by the United States Food and Drug Administration. On being intravenously injected, ICG rapidly binds to plasma proteins, especially lipoproteins, with minimal leakage into the interstitium. An absorption peak at 780 nm and emission peak at 830 nm were observed. [8] The absorption wavelength of water and haemoglobin containing an abundance of living tissue was <600 nm; thus, it was possible to excite the ICG. [9] Ex vivo indocyanine green detectability test Bovine serum albumin (BSA) powder was purchased from FUJIFILM Wako Pure Chemical (Osaka, JAPAN) and dissolved in distilled water to achieve concentrations of 5 × 10 −4 -5 × 10 −1 mg/mL. The BSA solution was added to the dye solution for the mixed solutions, and the mixtures were serially diluted in BSA solution to achieve 50-to 50,000-fold dilutions. To determine the optimal dilution for fluorescence detection, 100 µL of each dilution was added to individual wells in a 96-well plate and the plate was examined using the LFS system. This study was repeated thrice. Light emission detection by the LFS system was defined by a diphasic spectral graph with two peaks at approximately 780 nm. Our novel LFS system and a NIR camera system were used to emit excitation light from 10 mm away.

Indocyanine green detectability test with cadaveric porcine stomach
Endoscopic clips (model HX-610-135) were purchased from Olympus Medical System Corp. The front part of each clip was coated with BSA-conjugated ICG (5.0 × 10 −2 mg/mL; the brightest concentration in ex vivo ICG detectability test) [ Figure 2]. The distance was measured between the LFS system/NIR camera system and Figure 1: Novel laparoscopic near-infrared fluorescence spectrum system the clip positioned at the mucosal surface of the stomach wall. Our novel LFS system and the NIR camera system were used to emit excitation light from 10 cm away.

RESULTS
Serial dilutions of ICG conjugated to 0.05-0.75 g/mL BSA were imaged using the LFS system and NIR camera system. The concentration of 5.0 × 10 −2 mg/mL was brightest on the NIR camera system. We could not visualise fluorescence concentrations of <2.5 × 10 −3 mg/mL using the NIR camera system. However, the spectrum was detected the concentration of <2.5 × 10 −3 mg/mL on the LFS system [ Figure 3]. In the ICG detectability test with cadaveric porcine stomach, we were able to identify the spectrum of ICG (829 nm) to a 13-mm depth of the cadaveric porcine stomach wall using the LFS system [ Figure 4] but not with the NIR camera system regardless of wall thickness.

FIGS is rapidly emerging as a complementary technique
to conventional white-light reflectance imaging. Although several devices for FIGS have been described to date, only a few have progressed to clinical application. ICG is a popular diagnostic reagent that was approved clinically for the examination of hepatic and circulatory function. [10] The injected ICG binds rapidly to albumin and is carried more specifically through the vessels than indigo carmine or Evans blue. [11] ICG-related allergic reactions are less common than those of blue dyes such as isosulfan blue. [12] ICG has an absorption peak of 800 nm in vivo and is detected as a green colour. Detection by absorption spectroscopy is more sensitive than colour perception, and some surgeons reported that FIGS with ICG injection is useful for SLN detection. [13,14] In terms of ICG dye method limitations, such the loss of visibility in dense fat and rapid transit, some novel ICG-based techniques such as FIGS and ICG fluorescence imaging have been reported as convenient and reliable detection methods. However, we sometimes are unable to detect SLN using the NIR camera system due to a lack of clear delineation between the SLN and the surrounding fat tissue. Ishikawa et al. [13] reported an obese patient with a false-negative SLN by laparoscopic SLN navigation using the NIR fluorescence laparoscopic system despite infrared rays being able to penetrate fatty tissue up to a depth of 3 mm. Kitai et al. [15] noted in their preliminary report of ICG fluorescence imaging in breast cancer surgery that the sensitivity of fluorescence spectroscopy is much greater than that of absorption spectroscopy. They also reported that fluorescence was observed from an ICG solution embedded 1-cm deep in the material with optical properties compatible with human tissue in a preliminary study using a phantom. However, these systems have some limitations, including that the infrared rays cannot detect tumours within thick tissue or low concentrations of the Figure 3: Ex vivo indocyanine green detectability test. (a) Near-infrared system; (b) novel laparoscopic near-infrared fluorescence spectrum system. Serial dilutions of indocyanine green conjugated to 5-75 mg/mL bovine serum albumin were imaged using the laparoscopic fluorescence spectrum system and a near-infrared camera system. We could not visualize the fluorescence at concentrations of <5.0 × 10 −3 mg/mL using the near-infrared camera system but were able to confirm fluorescence concentrations of <2.5 × 10 −3 mg/mL in the laparoscopic fluorescence spectrum system b a fluorescent agent. Therefore, we developed the LFS system that detects the fluorescence spectrum as a solution to these problems. [5] Spectrum analysis is one method to overcome the imprecision that results from a subjective assessment. It is a method that consists of separating fluorescent light into a spectrum and measuring the peak fluorescence value. The novel LFS system enable to measure the fluorescent light spectrum laparoscopically, which also makes it possible to detect the weak fluorescent light of ICG for the invisible lesion. Here, we validated the usefulness of a novel LFS system in ex vivo and cadaveric porcine stomach models. We found that the novel LFS system successfully overcomes the limitations of NIR FIGS.

CONCLUSIONS
The novel LFS system successfully detected ICG on the NIR fluorescence spectrum. Our findings suggest that the LFS system can be integrated and routinely used in minimally invasive surgery.

Figure 4:
Indocyanine green detectability test with cadaveric porcine stomach. We were able to identify the spectrum of indocyanine green (829 nm) to a 13-mm depth of the cadaveric porcine stomach wall using the laparoscopic near-infrared fluorescence spectrum system but were unable to identify it with the near-infrared camera system regardless of wall thickness. Negative control: Non-fluorescence-coated clip