The monitoring of NADH levels in the tissue provides information on the metabolic state of the mitochondria in terms of energy production and intracellular oxygen levels [20]. NADH absorbs ultraviolet (UV) light. NADH is excited by UV light with wavelength of 320–380 nm and emits fluorescence with wavelength of 420–480 nm. NADH is a substrate or a coenzyme for the enzymatic activity of dehydrogenases that form part of respiratory chain and reside in the inner membrane of the mitochondria. Increase in the fluorescence intensity indicates a more reduced state of the NADH. Raji et al. measured NADH fluorescence to monitor injury level of myocardial infarction [21].

Another type of sources of fluorescent molecules used in surgical navigation is exogenous molecules introduced to the body, for example, by means of intravenous injection. Indocyanine green (ICG) is a near-infrared (NIR) fluorescent dye. ICG is FDA-approved, hydrophilic, anionic, tricarbocyanine near-infrared dye. It has the maximum absorption wavelength exists around 800 nm (765 nm [22], 800 nm[23]) and a molecular weight of 774.97 Da. Its safety is well validated in biomedical application so that it is easy to use in ICG for fluorescence surgical navigation [23, 24]. The fluorescence maximum of ICG is observed at 830 nm in blood [22]. Quantum yield of ICG in blood is higher than in H₂O. ICG has been widely used to visualize vasculature structure to evaluate blood perfusion in tissue. ICG was used to produce angiogram of retina [23]. ICG was introduced intravenously. A fundus camera with optical separator was used to take images of fundus. Unno et al. used ICG (0.5 %, 1 ml) to visualize vasculature. ICG was introduced intravenously to the patients who underwent paramalleolar artery bypass using saphenous vein grafts. ICG angiography could be obtained using a near-infrared camera in real time. Graft failure identified due to thrombotic occlusion after revision surgery was detected as portion without fluorescence [25]. Figure 44.4 shows the coronary circulation network of a porcine heart visualized by intravenously injected ICG. The heart was illuminated by infrared light from a semiconductor laser diode with emission wavelength of 790 nm with maximum power of 3.5 W. An infrared camera (electron multiplying CCD camera, ADT-100, Flovel, Tokyo Japan) with band-pass filter (813–850 nm) was used to obtain the images. The image shown in Fig. 44.4a was taken when the fluorescence intensity at coronary artery reached approximately the maximum value. As time elapsed, ICG moved to periphery resulting in increase of fluorescence intensity in entire myocardium. Then ICG moved to coronary vein. Figure 44.4b shows ICG image after artificial coronary artery was artificially ligated to generate temporally coronary artery occlusion. Ischemic area of myocardium was clearly visualized in real time.

ICG is also used to visualize sentinel lymph node during tumor resection procedure. Kitai et al. used intraoperative ICG fluorescence imaging to assess lymph node status in breast cancer. Light-emitting diode (LED) array that emits 760 nm light was used as an excitation light source. A charge-coupled device (CDC) camera equipped with a long pass filter with cutoff wavelength of 820 nm was used. 25 mg/5 ml ICG was injected in the subareolar breast tissue. They observed subcutaneous lymphatic drainage before and after skin incision. Lymphatic node could be clearly identified as a spot with fluorescence. Using this fluorescence image guidance, they resected fluorescent spots. All fluorescent nodes were regarded as sentinel lymph nodes [26]. Gotoh et al. applied the technique to identify hepatocarcinoma (HCC) nodules that were not detected by use of any preoperative examinations including intraoperative ultrasound imaging [27].

ICG fluorescence imaging is based on physical flow of ICG in anatomical structure and its characteristics of transport, storage, and secretion from the body. This is not directly related to the metabolic properties of pathological tissues. Fluorescence signals do not always represent tissue malignancy. This is the limitation of ICG-based fluorescence image surgical navigation.

Protoporphyrin IX (PpIX) serves as a prosthetic group in hemoproteins such as hemoglobin, myoglobin, and redox cytochrome. PpIX plays an important role in the regulation of protein synthesis and cell differentiation [28]. PpIX is a fluorescent substance excited by blue light with wavelength around 405 nm and emits red fluorescence around 635 nm. Orally administered 5-aminolevulinic acid (5-ALA) accumulates in tumor cells and is converted to PpIX by heme biosynthesis.

Stummer et al. used a fluorescence microscope to examine PpIX fluorescence for intraoperative normal tumor detection [29]. They reported that some region of the normal brain and tumor have different fluorescent characteristics. They also conducted a randomized controlled multicenter phase III trial of fluorescence-guided surgery with 5-ALA for resecting malignant glioma [30]. 5-ALA fluorescence-guided resection resulted an increase in the number of patients without residual and contrast-enhancing tumor on early MRI inspection compared with conventional surgical procedures under white light observation. Progression-free survival after 6 months was twice for fluorescence-guided resection group in comparison with the control group. Accumulation of 5-ALA in a brain tumor cell may be caused by a damaged blood–brain barrier (BBB) or enzymatic defects in the biosynthesis process shown in Fig. 44.5. So far, complete understanding on how PpIX accumulates in tumor cell has not been obtained although considerable information on the mechanism has been collected. Morphological and environmental such as tissue pH, enzymatic, and cell-specific factors should be considered comprehensively [28].