Indocyanine green is a small water-soluble molecule with molecular weight of 744.96 Da. ICG is excited at the wavelength of about 780 nm, and it emits fluorescence in the 700- to 850-nm range, which is not visible to the naked eye. After IV administration, ICG binds to plasma proteins and is cleared by the liver. In brain tumor surgery studies, 5- to 25-mg ICG concentrations were used (79, 80), and the observed duration of fluorescence was limited, with a peak at about 10 min.

Indocyanine green video angiography is a widely used method for intraoperative assessment of blood flow and vessel patency in tissue flap pedicles (81) and for assessment of intestinal perfusion near an anastomosis (82). ICG has been extensively studied for detection of sentinel lymph nodes in gastrointestinal oncology. Lymphatic drainage has been traced after subcutaneous ICG administration (83).

Although we do not discuss ICG angiography for vascular neurosurgery here, assessment of arterial and venous anatomy during some tumor resections may be necessary (84). Confirmation of the distal circulation with ICG angiography and test occlusion may be used when arterial sacrifice is required during tumor removal (85).

During a glial tumor resection using the operating microscope, ICG injection shows increased blood flow in the tumor tissue and pathology-induced alteration in the surrounding brain circulation (86). Hansen initially showed that ICG was able to highlight glioma tissue using an in vivo rat model (87), but therapeutically adequate doses did not produce sufficient fluorescence and required enhanced imaging technologies beyond the standard operating microscope (88, 89). Prior administration of bradykinin analog reportedly increased ICG extravasation and staining of tumor tissue in a glioma model (90) but not in the clinical setting. Simultaneous application of ICG during 5-ALA-guided glioma resection permitted detection of hypervascularized, angiogenic hotspots at the edge of resection potentially increasing the extent of resection (67). Although ICG produces an NIR signal with deeper penetration, it may not be specific for glial tumor tissue. ICG follows proteins leaked from the disrupted BBB and may diffuse into surrounding tissues. A unique liposomal formulated phospholipid-conjugated ICG has a particular brain-to-tumor biodistribution that may allow more accurate imaging guidance during surgery than ICG alone (91). Using a hand-held confocal endomicroscope, we observed that ICG selectively stained glioma cells in vivo (66).

Hemangioblastomas are highly vascularized tumors, and ICG fluorescence helps to identify hidden arterial feeders and vessels en passage (80, 92, 93). ICG has shown some usefulness in meningioma surgery. In cases of an occluded superior sagittal sinus, ICG was helpful in guiding the dural opening, tumor resection, and venous management, although multiple ICG injections were necessary (94). Additionally, ICG was used to highlight pituitary adenoma tissue through a microsurgical approach using the operative microscope (95, 96). ICG imaging using an endoscope was also recently reported to assist in visualization of tumors that infiltrated the sphenoid sinus (97) and to assess blood flow to the optic nerves and normal pituitary tissue during transsphenoidal surgery (98). ICG has also been used to guide transventricular endoscopic biopsies but not all areas of tumor dissemination were visible (99). ICG was further applied to visualize the facial nerve during temporal bone resection (100). After the facial canal was drilled to make it thin, ICG was injected and the fluorescence from the nerve blood supply guided further bony dissection to the internal acoustic meatus. Thus, the technique allowed visualization and preservation of the facial nerve (100).

One of the drawbacks of ICG visualization is that the image can only be displayed on a monitor, and technical refinements are needed to increase the comfort and ergonomics of ICG imaging instrumentation. Recent advances in this method include an overlay of fluorescence video angiography with a white-light field transmitted from the conventional operating microscope (101).

5-Aminolevulinic acid is a drug that is an intermediate metabolite of the heme synthesis pathway. 5-ALA is converted to protoporphyrin IX (PpIX), which is an endogenous fluorophore. PpIX peaks in 6 h after 5-ALA administration (102). Established correlation of gadolinium, a marker of BBB breakdown, with PpIX concentrations in glioma tissues, suggests (103) that BBB disruption is the leading cause of increased 5-ALA accumulation in malignant cells. However, increased 5-ALA-induced PpIX fluorescence was demonstrated within the areas with preserved BBB (104). PpIX has an excitation peak in the violet–blue light range (405 nm). Under blue light illumination, normal brain tissue reflects the light, whereas tumor tissue with accumulated PpIX emits a bright red fluorescence with two peaks, a large peak at 635 nm and a small peak at 710 nm.

Interest in 5-ALA application in neuro-oncology has been stimulated by promising PDT results with 5-ALA as the photosensitizer for the treatment of other types of cancers. PDT is recognized as a treatment modality mainly for tumors of hollow organs such as the stomach, colon, rectum (105), and most successfully, for skin malignancies (106). Its success is mainly related to a tumor location close to the surface that allows for sufficient depth of penetration by the irradiation.

For wide-field fluorescence, 5-ALA is usually administered 3 h before surgery so that the peak of PpIX production corresponds to the intraoperative tumor removal stage. Fluorescence observed in glioblastomas is often patchy and varies in intensity. Low-grade gliomas may not be visualized with wide-field techniques, although confocal endomicroscopy may detect 5-ALA in such tumors (107). In meningiomas, the observed fluorescence is usually high in intensity and is homogeneous (108). Tumor-specific fluorescence suffers from photobleaching. Natural fading occurs about 9 h after administration. 5-ALA-induced fluorescence decays to 36% of the peak within 25 min in light filtered to match the excitation wavelength of 405 nm; in contrast, with unfiltered wide-field illumination, 87 min was required to reach the same level of decay (25).

Most studies on glioma surgery with 5-ALA fluorescence for guidance have documented increases in tumor resection area (109, 110). The results of a phase III study indicated a 1.5-month increase in progression-free survival with 5-ALA fluorescence-guided surgery (111). In patients older than 55 years, regardless of tumor location, progression-free survival increased an additional 6 months. 5-ALA was also successfully used in brain tumor biopsy to obtain specimens of higher quality and to make a preliminary photodynamic diagnosis in a situation of primary central nervous system lymphoma (112).

Several approaches have advanced 5-ALA technology. One approach is to calculate the severity of the malignancy based on the fluorescence intensity. The emission spectrum must be analyzed accurately to calculate the ratio of peak emission intensity to the reflected excitation intensity (i.e., fluorescence intensity ratio). This ratio can then be used to predict the proliferative activity of the tumor (113). However, investigation of this characteristic was done in ex vivo tissue and requires technical improvement for intraoperative use. Other potential significant advancements for the use of 5-ALA involve the intraoperative use of high-magnification imaging optical technologies, such as confocal endomicroscopy, which may bring detection of fluorescence to the cellular level (107). The results of conventional histopathological methods correlated with confocal endomicroscopic imaging during 5-ALA-guided tumor resection (107). However, image quality was poor, and in vivo visualization of 5-ALA using blue laser confocal endomicroscopy in animal models could not confirm the findings (64, 107). Results from various clinical trials using intraoperative 5-ALA for brain tumor resection are ongoing (114).

5-Aminolevulinic acid, like all fluorophores, has drawbacks. Disruption of the BBB is necessary for fluorophore accumulation. In some low-grade gliomas, this may decrease or vary contrast accumulation. However, recent quantitative measurement studies suggest that diagnostic concentrations of PpIX do accumulate in low-grade tumors, but the concentration is below the detection threshold of current wide-field systems (115). Blood and overlying soft tissues can decrease visible fluorescence and hide the residual tumor. 5-ALA consumption and PpIX production may be highly variable (116) and depend on the several factors such as cell type (117), glucose concentration (118), pH (119), and other factors.

Fluorescein is an orange–red powder with the molecular formula C₂₀H₁₂O₅ and a molecular weight of 332.31 Da. It is widely used in the scientific and medical industries as fluorescein isothiocyanate 1 (FITC), Alexa 488 fluorophore, and other variants. In medicine, the fluorescein sodium salt is used, but for brevity, we refer to it here as fluorescein. Fluorescein as a marker of BBB disruption demonstrated perilesional edema in a cortical cold lesion model in rats (120). Tumor boundaries observed using fluorescein fluorescence correlate well with preoperative gadolinium contrast-enhanced boundaries (68). However, fluorescein has no particular interaction with the tumor cells and may not show fluorescence in diffuse, low-density tumor cell infiltrates (68, 121, 122).

Although the first clinical use of fluorescein for glioma surgery was in 1948 (20), fluorescein use in brain tumor surgery is not currently an FDA-approved use. Thus, it is restricted to clinical research studies. Its application is reported at several dosages. High doses of 15–20 mg/kg have been used for naked eye guidance without creating any permanent adverse effects (123). Yellow staining of the sclera, skin, and urine after high doses disappeared in approximately 24 h (124). Lower doses of 5–10 mg/kg for fluorescein-guided surgery using a special operative microscope module with excitation and observation filters were typically safe and effective in clinical trials (125), although one case of intraoperative anaphylaxis has been reported (126). The timing of fluorescein injection also varied across the studies. Some researchers have injected fluorescein intravenously after induction of anesthesia (127) at a dose of 3–4 mg/kg and waited for 10 min or 1 h, whereas others have injected it into a central venous line and waited for 20 min before resection (20, 124, 128–130). The half-life of fluorescein glucuronide, the main metabolite of fluorescein, is 264 min (131), and urinary clearance requires 24–32 h.

Fluorescein accumulates in glioma tissue homogenously and may be observed by the naked eye as bright to dark yellow staining of the tumor (123). Fluorescein-guided resection using operative microscopy without a special fluorescence detection module was reported by Shinoda et al. They achieved a gross total resection (GTR) in 84% of patients (129). Koc et al. produced GTR in 83% versus 55% of controls (132), and Chen et al. achieved an 80% GTR rate (123). The study by Koc et al. did not show a difference in survival (43.9 weeks in the patients given fluorescein and 41.8 weeks in the control patients) (132) while others did not assess survival.

A custom microscope for fluorescein-guided surgery was described in 1998 that increased fluorescent enhancement and contrast of intravenously injected fluorescein (8 mg/kg) during tumor removal (128), although there was diminished fluorescence in gadolinium unenhanced areas (65, 131, 133). The introduction of special filters to neurosurgical operative microscopes has stimulated interest in fluorescein-guided surgery, despite the dispute over fluorescein specificity for identifying tumor tissue.

Fluorescein has been used with success to guide removal of skull base tumors such as pituitary adenomas, craniopharyngiomas, meningiomas, and schwannomas (130). da Silva et al. reported enhancement of a meningioma dural tail by fluorescein (134). However, not all brain metastases and not all tumor areas were selectively highlighted by fluorescein, and some residual non-fluorescent tumor tissue was confirmed on postoperative enhanced MRI (135, 136).

Disruption of the BBB is an essential factor determining fluorescein extravasation, and several other factors may also confound fluorescein-guided glioma surgery. Variations in dose and timing of fluorescein administration may result in a variable degree of fluorescence in line with other factors such as fluorescein extravasation in surgically perturbed tissues, brain swelling, and unknown fluorescein distribution (127). Simultaneous administration of 5-ALA and fluorescein has shown that fluorescein was visible in normal brain and not detected in some areas highlighted by PpIX fluorescence (137). Thus, the benefit of fluorescein in guiding resection of malignancies is openly questioned and actively discussed. Some researchers warned that fluorescein application outside of clinical studies is premature and emphasized possible false positive and false negative staining during surgery (135). Nonetheless, confocal endomicroscopy with fluorescein in patients with brain tumors has revealed promising results on par with frozen section pathologic examination as a means of optically interrogating tissue (121).

Several new types of probes referred to as activity-based probes, “activatable” probes, fluorescence-quenched probes, or substrate-based probes were recently designed and investigated in preclinical studies (144–154) and recently reviewed in detail (155). Such probes contain a fluorophore that is “quenched” until the probe is activated (unquenched) by the given local environment. In caged-type fluorophores, some modified hydroxymethyl rhodamine green (Ac-HMRG; emission peak, 521 nm) probes are highly fluorescent when the quenching part of the probe is cleaved by its specific enzyme (57). One tumor-labeling strategy is to use quenching agents that are reconfigured by tumor-associated proteases, which are highly expressed in malignant tumor cells aiding invasion. For example, the matrix metalloproteinase-750 probe is activated by the broad range of matrix metalloproteinase family enzymes and facilitates accurate detection and complete removal of breast cancer tissue (58). One of the major drawbacks of untargeted probes, including activity-based probes, is their susceptibility to washout (active or passive removal from the site). One probe designed to be unquenched by cathepsin L and further covalently bound to protease reduced this limitation (152). Other limitations such as topical application of probes with inherent waiting time for binding, unknown biodistribution, and possibly uneven penetration await investigation. The practicality of fluorescence-guided surgery dictates that fluorescent tags should penetrate or bind to the cancer cells or remain in proximity to them for long enough to detect them.

One interesting approach is the design of activity-based probes such as caspase-sensitive nano-aggregation fluorescent probe (C-SNAF) that microaggregate after cleavage by caspase-3 and -7 by intramolecular cyclization (60). Another exciting approach is the complex activity-based probe. The construct is synthesized by combining a cell-penetrating peptide that may be activated and a nanoparticle labeled with gadolinium and Cy5 fluorophore. This complex probe is taken up by the tumor cells after matrix metalloproteinases 2 and 9 cleave the peptide and activate its cell-penetrating domain. In a murine model, gadolinium allowed in vivo visualization of the tumor using MRI and Cy5 allowed fluorescence detection (156). The advantages of such probes are the ability to carry several labels for various types of detection, high contrast to the background, and applicability to a wide range of tumors. However, this model was not tested for application in brain tumors.

Molecular targeted probes are also known as affinity-based probes. Targeting molecules with colored and fluorescent dyes has revolutionized microscopy. Application of this method of visual guidance for tumor resection is under investigation in cell cultures and animals by several research groups (149–154, 157, 158). Many known tumor targets such as EGFR, HER2, CD105, VEGFR, and folate receptors have been tested for fluorescence visualization of tumors. Additionally, targeting and highlighting of normal peripheral nerves have been investigated to prevent nerve injury during surgery (159).

Molecular targeted probes may be classified based on the fluorophore, targeted molecule, and other components. The majority of the targeting molecules fall into three categories:

Antibodies;

The rapid growth of targeted molecular probes has occurred because of the development of new fluorophores that may be conjugated to a variety of specific targeting molecules. Numerous possible combinations, including the possibility of adding a second (or more) label, significantly increase this potential. Many fluorophores have become commercially available and are being investigated in numerous preclinical and several clinical trials. Two clinical trials of IRD 800CW-labeled probes for visualization of breast cancer and familial adenomatous polyposis have been completed (164, 165), and other trials are recruiting patients (166–172) for the use of the Cy 5.5-labeled probe (173). However, none of these trials involve brain tumors. A promising fluorescent label, zwitterionic NIR fluorophore, ZW800-1, was recently described (50). ZW800-1 has great promise as it shows a higher tumor-to-background ratio than IRDye800-CW and Cy5.5 in vitro and in vivo (17.2 vs. 5.1 and 2.7, respectively) (50, 51).

The major drawbacks of targeted molecular probes are uneven passive distribution and non-specific binding. Dual-labeled probes were designed to address this limitation (174). This approach uses targeted probes that bind, and untargeted probes that do not bind, to the target. Differentiation in the fluorescence intensity allows a quantitative assessment of the binding potential of the probe (175, 176). Another drawback is that tumor regions with an undisrupted BBB can decrease the accumulation of the large probes. Sexton et al. addressed this problem by designing a small targeted fluorescent affibody peptide (about 7 kDa vs. 150 kDa for a full antibody) and demonstrated in a mouse xenograft GBM model almost two times increased fluorescence at the tumor edge compared to the full anti-EGFR antibody probe (42). Gong et al. showed that both anti-EGFR-specific affibody and the therapeutic antibody panitumumab labeled with IRDye 800CW could be used as imaging agents for both wild-type EGFR and EGFRvIII glioblastoma cells in cell culture studies (177).

In a 2014 report, Ghosh et al. described a novel targeted probe construct containing a single-walled carbon nanotube as a fluorescent tag (52). It consists of an M13 bacteriophage as a scaffold, a targeting protein, and the fluorescent nanotube. The single-walled carbon nanotubes have emission within the NIR range (950–1400 nm), resulting in less optical scattering and deeper tissue penetration. This setup is also less susceptible to photobleaching or quenching effects. The construct was stable and showed 10 times more selective fluorescent staining of ovarian tumor cells than the same construct without the targeting peptide. The nanotube fluorescence intensity ratio relative to the background (5.5 ± 1.2) was superior to the same construct labeled with other NIR AlexaFluor750 dye (3.1 ± 0.42) or FITC (0.96 ± 0.10). However, this study did not assess the possible penetration of the probe into the brain (52).

A targeted probe consisting of fluorescent gold nanoparticles conjugated with diatrizoic acid and AS1411 aptamer (53) has an absorption band of 300–400 nm and orange–red emission (maximum 620 nm), which can be observed by the naked eye. This probe showed specific binding to tumor cells due to the AS1411 aptamer, which targets nucleolin. The probe allowed X-ray visualization due to the high electron density of the gold nanoparticles. A novel lymphoma-specific fluorescent (Alex488) switchable TD05 aptamer in a human brain tumor xenograft model has been described (54). This probe rapidly and precisely identified human B cell lymphoma in biopsies. Such a system would be useful for rapidly discriminating non-operative CNS B-cell lymphoma from malignant glioma based on the biopsy.

Another agent, BLZ-100, a tumor ligand chlorotoxin conjugated to ICG, was shown to have high affinity to human gliomas in mice (55). Chlorotoxin was extensively studied in preclinical in vivo studies as a conjugate with Cy 5.5 (178) and IRDye 800CW (179). Chlorotoxin is a new drug that binds to chloride channel–MMP-2 membrane complexes. It reduced the invasiveness of glioma cells (180), inhibited glioma cell growth and metastasis, and accelerated tumor apoptosis (181). The advantage of the probe is its small size and ability to penetrate the BBB.

Another 5-carboxyfluorescein-labeled fluorescent probe consisting of tLyP-1 small peptide targeting neuropilin receptors was recently described. Neuropilin receptors are co-receptors for vascular endothelial growth factor and play a role in tumor-mediated angiogenesis. They are overexpressed in most gliomas. The probe has selective uptake and may have advantages over the CTX-Cy5.5 probe due to its small size. However, the fluorescein labeling was less than ideal and could be exchanged for a more intense fluorophore for use in intraoperative imaging (56).

IRDye800CW-labeled anti-EGFR nanobody 7D12 was compared to the full antibody cetuximab and showed better penetration and distribution of the nanobody probe in vivo in a preclinical study (40). Another study of the same nanobody for orthotopic tongue tumors showed significantly higher tumor to background fluorescence (2.00 ± 0.34 in the FLARE imaging system) than in the group with the same non-targeted fluorescent probe (41).

Polyacrylamide nanoparticles have been coated with the F3 protein that binds to nucleolin and loaded with methylene blue, Coomassie blue, or ICG. F3-coated constructs increased the color change in glioma cells in vitro (61). Magnetic NH₂-cross-linked iron oxide nanoparticles labeled with Cy5.5 (32 nm in diameter) produced clear tumor border demarcation and co-localization on MRI imaging in a rat gliosarcoma model (62). As a non-targeted construct, it produced demarcation mainly due to BBB disruption and was designed as a magneto-optical probe. Additionally, iron oxide particles are eliminated by reticuloendothelial cell endocytosis (182), suggesting that their elimination is more predictable than that of other nanoprobes. However, this magnetic nanoparticle design is less attractive than that of targeted probes, which aim to increase the accuracy of tumor cell visualization in vivo.

Summarizing fluorophore use in neurosurgery, 5-ALA-guided brain tumor surgery may improve the gross tumor resection rate and is approved in Europe but is available only in clinical trials in the US. Fluorescein-guided resection has emerged as an alternative due to its safety profile, although fluorescein is not tumor cell-specific. ICG shows promise for vascular tumors, such as hemangioblastomas, but may also have the potential to define malignant gliomas. Many new targeted and activatable fluorescent probes are awaiting full assessment to be used in clinical studies. Although molecular targeting probes are attractive and technologically advanced, their benefit and cost compared to already existing 5-ALA and fluorescein for fluorescence-guided resection are yet to be proven. Assessing the advantages of the many probes being designed is a difficult and time-consuming task considering the emerging improved, quantitative fluorescent detection methods. Combining the probes with molecules for secondary goals such as chemotherapy, photosensitization, and others may be advantageous.

The use of custom operative microscopes with modules to measure fluorescein (128) and PpIX (190) fluorescence was reported in 1998. There are three fluorescence detection modules, for use on commercially available operative microscopes for 5-ALA- (Figure 2), ICG- (Figure 3), and fluorescein-guided (Figure 4) tumor resection in the brain. The modules consist of three components: (1) a set of optical filters for selective wavelength separation, (2) a broad-spectrum illumination device, and (3) optional CCD cameras for detection of visible and invisible (NIR) light (Figure 3). New optical filters for selection of bandpass width and blocking intensity are now available due to progress in optical engineering. A combination of these new filters permits the best possible fluorescence intensity and contrast to the background ratio. One example is the new Yellow 560 Module for the Zeiss operative microscope that reintroduced fluorescein in brain tumor surgery. A set of filters facilitates excitation of the fluorescein with the maximum intensity while preserving illumination of the background with another visible spectral band of lower intensity (Figure 4). The resulting bright yellow fluorescence of tumors is observed and contrasted with the natural colored background (191).

Fluorophores that emit in the NIR spectrum require CCD cameras or other detection technologies. An ICG (NIR) module does not require an operative microscope per se because the resultant fluorescence is not perceived by the eye and is observed on an ancillary screen. The fact that NIR probes are in abundance and are either commercially available or awaiting approval by the FDA (ICG, Cy 5.5, IRDye800-CW, and BLZ-100) has stimulated the field of computer engineering to develop wide-field systems that overlay an NIR signal on the surgical field of view. Such an overlay is desirable in real time and with measurable specificity. Non-microscopic, mobile, wide-field video imaging systems for open, laparoscopic, thoracoscopic, and robotic surgery are in development and clinical trials (192, 193).

Work in intraoperative NIR imaging technologies in neurosurgery shows potential for advantageous applications. A novel proof-of-concept NIR imaging system consists of a narrow-band laser at 785 nm, a notch filter, and a standard 2-CCD camera for wide-field visualization. This system has been tested with an ICG-conjugated targeted BLZ-100 probe in a murine brain tumor model (55).

A new endoscopic technology, scanning fiber endoscopy (194), also holds great promise due to its ultrathin probe and increased resolution. A color image is acquired by combining red, blue, and green laser lights through a spiral actuated optical fiber. Laser induced fluorescent imaging of 5-ALA-induced PpIX fluorescence on tumor cell phantoms (195) and a murine tumor model (work in progress) allowed detection of the fluorescence with greater sensitivity than through the operative microscope. We found this optical imaging technology very convenient for potential intraoperative use due to its small size with a field of view of 2–30 mm and high spatial resolution of up to 15 μm.

A concept for a low-cost fluorescein detection system for glioma surgery (196) consists of a xenon light source, fiber optic light cable, a set of glass interference filters (neutral, 490 nm, 465 nm), and yellow photographic filters for oculars or UV yellow glasses. In clinical trials, the system showed great potential due to its low cost, especially beneficial for low-income countries, although limitations of the custom hardware and fluorescein usefulness in glioma surgery itself still require confirmation (127).

Limitations of wide-field visualization technologies in fluorescein-guided surgery are similar to those of 5-ALA studies. Wide-field, fluorescence-guided surgery limitations include (184) ambiguity at the margins where fluorescence intensity decays and difficulty of visualization on the sides of a resection cavity and shaded areas in the surgical wound. Further limitations include fluorescence absorbance by blood and tissue layers (197), insufficient fluorescence intensity in >95% of low-grade gliomas (107, 198, 199), and lack of quantitative assessment of fluorescence intensity. In PpIX fluorescence visualization, PpIX is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas (117). Such works emphasize the limitations of fluorescence detection by the current wide-field technologies at low concentrations of the fluorophore or when few cells are labeled. New approaches that increase the sensitivity of visualization systems include the use of a quantitative spectrophotometer, an additional camera for quantitative image processing (200), and new endoscopic and confocal endomicroscopy probes.