Edited by: Eberval Figueiredo, Universidade de São Paulo, Brazil
Reviewed by: Yasunori Fujimoto, Osaka University, Japan; Andrei Fernandes Joaquim, University of Campinas, Brazil
Specialty section: This article was submitted to Neurosurgery, a section of the journal Frontiers in Surgery
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Fluorescence-guided surgery is one of the rapidly emerging methods of surgical “theranostics.” In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients as well as future applications of recent laboratory and translational studies.
Review of the literature.
A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence-guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-aminolevulinic acid, and indocyanine green), less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine, can be used for rapid tumor detection and pathological tissue examination. Other emerging agents, such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment, are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed.
We are standing on the threshold of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.
Malignant glioma is a highly invasive, heterogeneous, complex, and fatal tumor type, the extent of which is not precisely identifiable by modern imaging techniques. Despite all of the current treatment modalities for malignant gliomas, such as microsurgery, chemotherapy, and radiotherapy, there is no definitive treatment. Nonetheless, the maximum extent of surgical resection is associated with a longer recurrence-free period and overall survival of patients with glioblastomas (
Advances in imaging began with the philosophies of cerebral localization and function, while techniques for improving precision and the customization of brain tumor surgery can be traced to the late nineteenth century. The evolution of imaging techniques in neurosurgery began with the first attempts at craniometric localization of intracranial lesions (
Techniques for visually identifying the tumor mass began in the mid-twentieth century. The application of the fluorescent dye fluorescein sodium to highlight tumor tissue during its removal was introduced in neurosurgery by Moore et al. in 1948 (
Accurate visualization of brain tumors marked by fluorescent probes and even residual tumor cells is possible with emerging new technologies. These emerging technologies are expected to become state-of-the-art tools to maximize customized brain tumor treatment. These technologies are the logical extension of the evolution of the search for precision in brain tumor surgery. Such technologies will allow real-time imaging interrogation of the brain during surgery at the cellular resolution to maximize or tailor brain tumor resection.
This review summarizes recent achievements and future perspectives of clinical, laboratory, and translational studies that bring fluorescence-guided neurosurgery to the cellular level, thereby allowing for individualized brain tumor resections, representing a crucial breakthrough in this field.
In the last decade (2006–2016), the number of fluorescent stains and cellular tags used in preclinical studies has increased significantly, with many novel fluorophores awaiting approval for clinical use. The fluorescent probes and dyes discussed in this review are summarized in Table Passive fluorescent probes (ICG, fluorescein sodium, and other stains); Metabolic probes (5-ALA, activatable probes); and Targeted probes.
Name of probe | Reported excitation wavelength | Reported reading emission wavelength | Used equipment | Species tested | Advantages | Disadvantages | Mode of administration and time to imaging (unless noted otherwise) |
---|---|---|---|---|---|---|---|
IRDye 800CW-labeled VEGF ( |
675 and 745 nm ( |
800 nm ( |
IVIS Spectrum (PerkinElmer, Inc.) Multispectral Fluorescence Camera System (Institute for Biological and Medical Imaging, Technical University, Munich, Germany and SurgOptix Inc., Redwood Shores, CA, USA), Olympus Fluoview 300 Confocal Scan Box mounted on an Olympus IX 71 inverted microscope (Olympus America Inc.), Pearl Imaging System (LI-COR Biosciences) |
Xenograft mice model (human ovarian, breast, and gastric cancers) | Distinguish submillimeter lesions intraoperatively. Longer lasting and more accurate signal for VEGF and EGFR2 than ICG alone. Bevacizumab-800CW fluorescence detection in extracellular matrix, trastuzumab-800CW fluorescence detection on tumor cell surface | Long half-life for detecting tumors. Long elimination time | IV, 6 days (optimal time) |
IRDye 800CW-labeled human EGFR 2 [Trastuzumab ( |
675 and 745 nm ( |
800 nm ( |
IVIS Spectrum (PerkinElmer, Inc.) Multispectral Fluorescence Camera System (Institute for Biological and Medical Imaging, Technical University and SurgOptix Inc.), Olympus Fluoview 300 Confocal Scan Box mounted on an Olympus IX 71 inverted microscope (Olympus America Inc.), Pearl Imaging System (LI-COR Biosciences) |
Xenograft mice model (human ovarian, breast, and gastric cancers); Xenograft mice model (human breast cancer lymph metastasis) | Distinguish submillimeter lesions intraoperatively. Longer lasting and more accurate signal for VEGF and EGFR2 than ICG alone. Bevacizumab-800CW fluorescence detection in extracellular matrix, trastuzumab-800CW fluorescence detection on tumor cell surface | Long half-life for detecting tumors. Long elimination time | IV, 3–6 days (optimal time); 3 h for lymph node visualization |
IRDye 800CW-labeled anti-EGFR nanobody 7D12 ( |
760 nm; 656–678 nm; 745–779 nm | 774 nm; 700 nm; 800 nm | IVIS Lumina System (PerkinElmer, Inc.) with ICG filter sets FLARE imaging system (Beth Israel Deaconess Medical Center) IVIS Spectrum (PerkinElmer, Inc.) |
Xenograft mice (human epidermoid carcinoma); xenograft mice (human metastatic oral squamous cell carcinoma) | Better tumor penetration and distribution of nanobody probe |
Not mentioned | IV, 30 min (earliest); 2 h (optimal); or 24 h (optimal) |
IRDye 680RD labeled EGFR inhibitor (cetuximab) ( |
620 nm | 650–800 nm | Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, Nebraska), |
Xenograft mice (human U251 glioma) | Higher affinity for tumor than anti-EGFR targeted affibody used in same study | Concentration of antibody in tumor focused primarily in the center | IV, 1 h |
IRDye 800CW-labeled anti-EGFR targeted affibody ( |
720 nm | 730–900 nm | Odyssey Infrared Imaging System (LI-COR Biosciences), |
Xenograft mice (human U251 glioma) | Smaller size molecule results in better penetration of BBB. Higher concentration in outer tumor than antibody | 30 times lower affinity than antibody and a shorter plasma half-life | IV, 1 h |
IRDye 800CW-labeled chemokine stromal cell derived factor-1 (SDF-1) ( |
685 and 785 nm | 702 or 789 nm | Pearl Imaging System (LI-COR Biosciences), |
Xenograft mice (A764 human glioma, MCF-7 human breast cancer) | Detected as low as 500 cells |
Labeled bone marrow, transient non-specific labeling during first 24 h was observed in the liver and skull | IV, 1-h visualization of tumors and background structures; 24–92 h background fluorescence diminished, tumors remained clearly visible |
IRDye 800CW-labeled anti-CD105 monoclonal antibody (angiogenesis related) ( |
778 nm | 806 nm | Pearl Imaging System (LI-COR Biosciences) |
Mice with 4T1 mouse breast cancer; human MCF-7 breast cancer cells in cultures | Tumor could be visualized as early as 30 min post-injection; may be used in the clinic for imaging tumor angiogenesis | CD105 expression is observed only on actively proliferating tumor endothelial cells | IV, 30 min (early); 16 h (optimal) |
Cy5.5-labeled EGFR inhibitor (cetuximab) ( |
683 nm (max); 630–670 nm (range used in experiment) | 707 nm (max); 685–735 nm (range used in experiment) | Leica MZFL3 stereo research microscope (Leica Microsystems, Bannockburn, IL, USA) fitted with a GFP and Cy5.5 filter and an ORCA ER charge-coupled device camera (Hamamatsu, Bridgewater, NJ, USA) eXplore Optix time-domain fluorescence imaging system (ART/GE Healthcare, Princeton, NJ, USA) |
Cell cultures: UM-SCC-1, FaDu, CAL 27, and AB12; xenograft mice model (human head and neck squamous cell carcinoma cell lines SCC-1, FaDu, CAL 27); mice with mouse mesothelioma | Can be used to detect tumors |
EGFR expression did not correlate with the fluorescent intensity | IV, 48–72 h (optimal) |
Alexa-680 labeled insulin-like growth factor 1 receptor (IGF1 R) (AVE-1642-conjugated Alexa 680) ( |
575–605 nm | 645–850 nm | Maestro Imaging System (CRI), Olympus Fluoview FV500 laser scanning confocal system (Olympus America Inc.) |
Xenograft mice model (MCF-7 human breast cancer cells) | Can detect the downregulation of IGF1R after treatment with a monoclonal antibody | Further studies required to determine the amount of background fluorescence produced by IGF1R | 1 day (earliest); 2 days (clear imaging) |
Folate–fluorescein isothiocyanate probe (for folate receptor) ( |
495 nm | 520 nm | Intraoperative Multispectral Fluorescence Camera System (Institute for Biological and Medical Imaging, Technical University) | Humans with ovarian cancer | High specificity for labeling FR-alpha expressing cells. Real-time image-guided excision of fluorescent tumor deposits of size <1 mm was feasible | Four patients experienced mild discomfort in the upper abdominal region after injection | Imaging completed 2–8 h after injection |
BODIPY FL-labeled PARP inhibitor (Olaparib) ( |
503 nm | 515 nm | Maestro Imaging System (CRI) | Xenograft mice model (U87 MG and U251 MG human glioblastomas) | High specificity for the DNA repair enzyme PARP1 with therapeutic effect. Promising new targeted antitumor drug, which is already in clinical trials. High tumor-background fluorescent ratio. Toxicity profile is known and similar to Olaparib | Not mentioned | 60–180 min (optimal) |
Liposomes with RGD peptide and the neuropeptide SP, gadolinium, Indium-111, Rhodamine-B ( |
554 nm | 576 nm | Zeiss LSM 510 Microscope (Carl Zeiss Meditec AG, Jena, Germany) | Cultured mouse fibroblast cells with U87 MG human glioblastoma and M21 human melanoma tumor cells ( |
Combination of radioactive, fluorescent, and magnetic resonance imaging signaling; multifunctionality of liposomes as a carrier of different probes | Moderate tumor uptake | |
ZW800-1 zwitterionic NIR fluorophore ( |
750 ± 25 nm; 773 nm | 810 ± 20 nm; 790 nm | FLARE Imaging System (Beth Israel Deaconess Medical Center) FLARE Imaging System (Beth Israel Deaconess Medical Center) Pearl Small Animal Imaging System (LI-COR Biosciences) |
Xenograft mice model (M21 human melanoma, Lewis lung carcinoma, HT-29 human colorectal adenocarcinoma) | Higher tumor-to-background ratio than IRDye800-CW and Cy5.5 | Wash-out of dye from tumors started occurring at 4 h (dye still present at 24 h) | 4 h, low visibility at 4 h, highest visibility from 24 to 72 h |
M13-stabilized single-walled carbon nanotubes (SBP-M13-SWNTs) ( |
808 nm | 950–1400 nm | Liquid nitrogen-cooled OMA V 2D InGaAs array detector with a 256 × 320 pixel array (Princeton Instruments) coupled with SWIR-25 NIR camera lens (Navitar, Rochester, NY, USA) | Xenograft mice model (OVCAR8 human ovarian epithelial carcinoma) | Stable and showed 10 times more selective fluorescent staining of ovarian tumor cells than same construct without targeting peptide. Nanotube fluorescence intensity relative to background (5.5 ± 1.2) was superior to same construct labeled with other NIR AlexaFluor750 dye (3.1 ± 0.42) or FITC (0.96 ± 0.10) | Study did not assess possible penetration of the probe into the brain | 24 h |
Fluorescent gold nanoparticles conjugated with diatrizoic acid and AS1411 aptamer ( |
400 nm | 620 nm (max) | Ultra-VIEW RS Confocal System (PerkinElmer, Inc., Waltham, MA, USA) IVIS (PerkinElmer, Inc.) |
Xenograft mice model (human lung adenocarcinoma) separate MCF-7 cell assay | Specific binding to tumor cells due to AS1411 aptamer, which targets nucleolin. Allowed X-ray visualization due to high electron density of gold nanoparticles | Small sample size ( |
30 min |
Lymphoma-specific fluorescent (Alex488) switchable TD05 aptamer ( |
489 nm | 505–535 nm | Zeiss 710 laser Scanning Confocal Microscope (Carl Zeiss Meditec AG) equipped with a 40×/1.2NA water emersion objective ( |
Xenograft rat model (U251 human glioma and Ramos human CNS lymphoma) | Probe could rapidly and specifically identify human B cell lymphoma in biopsies. System would be useful for discriminating non-operative CNS B-cell lymphoma from malignant glioma rapidly after biopsy | Total antibody staining time was 24 h and aptamer staining time was 1 h ( |
|
Chlorotoxin (CTX) conjugated to ICG (BLZ-100) ( |
785 nm | Near-infrared spectrum | Custom imaging system: 16-mm VIS-NIR Compact Fixed Focal Length Lens (Edmund Optics, Barrington, NJ, USA) coupled 785-nm StopLine single-notch filter, NF03–785E-25 (Semrock, Rochester, NY, USA) | Xenograft mice model (LN229 human glioblastoma) | High affinity to human gliomas | Not mentioned | 48 h |
5-Carboxyfluorescein (FAM)-labeled fluorescent probe consisting of tLyP-1 small peptide targeted to the neuropilin receptors (FAM-tLyP-1) ( |
Blue light | Not given | Kodak |
Xenograft mice model (U87MG human glioblastoma) | Selective uptake. May have advantages over CTX-Cy5.5 probe due smaller size | Fluorescein labeling was less than ideal, could be exchanged for more intense fluorophore | 1 h |
Modified hydroxymethyl rhodamine green (gGlu-HMRG) ( |
488 nm | 505–530 nm | In-house-made portable fluorescence camera for Zeiss LSM510 Microscope (Carl Zeiss Meditec AG) |
Human breast cancer tissue samples; breast cancer cell culture | High sensitivity and spatial resolution |
In breast cancer, this method cannot distinguish malignant and benign regions | 5 min |
MMPSense 750 FAST (MMP-750) ( |
749 nm | 775 nm | Surgical Navigation System (Institute of Automation, Chinese Academy of Sciences, Beijing, China) (59) | Mice with 4T1-luc breast cancer tumors | Imaging method offered precise detection of the orthotopic breast tumors and metastases intraoperatively in real time | Not mentioned | IV, 6 h (fluorescent signal observed); 24–36 h (optimal fluorescent signal) |
Caspase-sensitive nano-aggregation fluorescent probe (C-SNAF) ( |
635 ± 25 nm | 670–900 nm | Maestro Hyperspectral Fluorescent Imaging System (CRI) | Xenograft mice model (subcutaneous HeLa tumors) | Highly feasible for imaging of drug-induced tumor apoptosis |
Not mentioned | IV, 1 h |
Polyacrylamide-based nanoparticles loaded with ICG or Coomassie blue dye ( |
647 nm | 675–725 nm | Olympus IX70 confocal microscope (Olympus America, Inc.) Ultra-VIEW Confocal Laser Scanning Microscope (PerkinElmer, Inc.) |
Cell cultures: 9L rat gliosarcoma, MDA-MB-435 human melanoma, MCF-7 human breast cancer | Produced visible color change in tumor cell lines | Significant non-specific binding was observed | Imaged after 2 h of incubation |
Iron oxide magnetic NH2-CLIO nanoparticles labeled with Cy5.5 (Cy5.5-CLIO) ( |
Not given | Not given | Custom-built surface reflectance imaging system (Siemens Medical Systems, Erlangen, Germany), Zeiss LSM 5 Pascal (Carl Zeiss Meditec AG, Jena, Germany), |
Rat 9L gliosarcoma tumor model | Clear tumor border demarcation |
Not as accurate as target probes for |
IV, 24 h |
Cyto647 labeled anti-EGFR antibody-conjugated SERS-tagged gold nanoparticles (antibody-Panitumumab) ( |
642 nm (Olympus); 785 nm (Raman) | 700–775 nm | Olympus IX81 inverted fluorescence microscope (Olympus America, Inc.) Hamamatsu Back-Thinned EM-CCD camera, 9100-13 (Hamamatsu, Bridgewater, NJ, USA) Spinning Disk Confocal Scanning Raman Microscope (Renishaw, Wotton-under-Edge, UK) |
Selective uptake by tumor cells; unlike other fluorescent dyes, SERS nanoparticles have enhanced photostability | Not mentioned | Not applicable | |
5-ALA that metabolically converts into fluorescent PpIX | 400–410 nm violet | 620–720 nm red | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical GmbH) |
Studies in human ( |
Studies have shown increased extent of tumor resection with PpXI guided surgery; useful for brain tumor biopsy | Disruption of BBB necessary for fluorophore accumulation (can decrease/vary contrast) |
Oral, IV, 2 h ( |
Indocyanine green (ICG) | 780 nm | >795 nm | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical, GmbH) Zeiss LSM710 (Carl Zeiss Surgical, GmbH) |
Mice with GL261 mouse glioma ( |
Extensively studied; hand-held confocal endomicroscope and LSM showed ICG selectively stained glioma cells in mouse model ( |
ICG visualization can only be displayed on a monitor | IV, 15 min |
Human ( |
Intraoperative administration at end of 5-ALA guided resection may show additional tumor tissue ( |
||||||
Fluorescein sodium ( |
494 nm | 521 nm | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical GmbH) LSM710 (Carl Zeiss Surgical, GmbH) |
Human | Convenience for surgeon, surrounding tissue has more natural color | Rapid photobleaching, non-specific accumulation of fluorescein along the margins of resection. Possible extravasation along with edema | IV, 5 min ( |
CLR1501 ( |
500 nm | 517 nm | Nikon A1RSi Confocal Microscope (Nikon, Minato, Tokyo, Japan); IVIS Spectrum system (PerkinElmer, Inc.) | Xenograft mouse model (U251 human glioblastoma, 22T, 22CSC, 33CSC, 105CSC patient derived glioblastoma) | Tumor-to-brain fluorescence ratio similar to 5-ALA | Tumor must be visualized on separate monitor | IV, >4 days |
CLR1502 ( |
760 nm | 778 nm | IVIS Spectrum system (PerkinElmer, Inc.) Fluobeam 800 (Fluoptics, Grenoble, France) Leica OH4 intraoperative microscope with FL800 attachment (Leica Microsystems, Bannockburn, IL, USA) |
Xenograft mouse model (U251 human glioblastoma, 22T, 22CSC, 33CSC, 105CSC patient derived glioblastoma) | Tumor-to-brain fluorescence ratio superior to 5-ALA | Tumor must be visualized on separate monitor | IV, >4 days |
CH1055 ( |
~750 nm | 1055 nm | In-house-built NIR spectroscopy instrument with Acton SP2300i spectrometer (Princeton Instruments, Trenton, NJ, USA) and Princeton OMA-V liquid-nitrogen-cooled InGaAs linear array detector (Princeton Instruments) | Xenograft mice model (U87MG human glioblastoma) | High tumor-to-background signal ratio |
Tumor must be visualized on separate monitor | IV, 6 h (tumor is clearly visible); 72 h (optimal) |
Acridine orange ( |
488 nm | 505–700 nm (LSM); 505–585 (VWCE) | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical GmbH) LSM710 (Carl Zeiss GmbH) |
Mice with GL261 glioma; swine normal brain | Suitable for rapid intraoperative |
Cannot be used in the brain due to toxicity profile | Topical application, immediately |
Acriflavine ( |
405 nm (LSM); 488 (VWCE) | 505–585 nm | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical GmbH) LSM710 (Carl Zeiss GmbH) |
Mice with GL261 glioma | Suitable for rapid intraoperative |
Cannot be used in the brain due to toxicity profile | Topical application, immediately |
Cresyl violet ( |
561 nm (LSM); 488 nm (VWCE) | 620–655 nm (LSM); 505–585 nm (VWCE) | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical GmbH) LSM710 (Carl Zeiss GmbH) |
Mice with GL261 glioma | Highlights tumor boundaries |
No current |
Topical application, 10 min |
Sulforhodamine 101SR101 ( |
561 nm (LSM); 488 nm (VWCE) | 585–615 nm (LSM); 505–750 nm (VWCE) | VWCE Zeiss Pentero Microscope (Carl Zeiss Surgical GmbH) LSM710 (Carl Zeiss GmbH) |
Xenograft rat model (U251 human glioma) | Strongly labeled cells within the tumor and astrocytes within normal brain | Non-specific | 1 h |
Demeclocycline ( |
402 nm | ~520 nm | Custom confocal laser scanning microscope | Human low- and high-grade glioma tissues | Highlights tumor cells |
Non-specific | Topical application, timing not reported |
Methylene blue ( |
642 nm | ~690 nm | Custom confocal laser scanning microscope | Human meningioma, glioma, and adenocarcinoma tissues | Highlights tumor cells |
Non-specific | Topical application, timing not reported |
One of the most important characteristics of the probes is their ability to accumulate in tumor tissues in high concentrations. In the case of brain tumors, the blood–brain barrier (BBB) influences the delivery of probes that are not lipophilic or have a molecular weight more than 400–600 kDa (
In this section, we discuss fluorescent agents that are used or could potentially be used for fluorescence-guided resection and intraoperative diagnosis of brain tumors.
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 (
Indocyanine green video angiography is a widely used method for intraoperative assessment of blood flow and vessel patency in tissue flap pedicles (
Although we do not discuss ICG angiography for vascular neurosurgery here, assessment of arterial and venous anatomy during some tumor resections may be necessary (
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 (
Hemangioblastomas are highly vascularized tumors, and ICG fluorescence helps to identify hidden arterial feeders and vessels en passage (
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 (
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 (
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 (
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 (
Most studies on glioma surgery with 5-ALA fluorescence for guidance have documented increases in tumor resection area (
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 (
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 (
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 (
Although the first clinical use of fluorescein for glioma surgery was in 1948 (
Fluorescein accumulates in glioma tissue homogenously and may be observed by the naked eye as bright to dark yellow staining of the tumor (
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 (
Fluorescein has been used with success to guide removal of skull base tumors such as pituitary adenomas, craniopharyngiomas, meningiomas, and schwannomas (
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 (
Various new fluorophores and smart-targeted fluorescent probes are in different stages of preclinical development. Here, we review new fluorescent labels and activity-based and targeted bioengineered fluorescent probes.
Cresyl violet, acridine orange, and acriflavine are fluorescent dyes that were investigated for
Demeclocycline (excitation/emission peaks at 458/529 nm) is a tetracycline antibiotic with phototoxic effects. It has been used to demarcate tumor cells when used as an
Novel cancer-selective alkylphosphocholine analog fluorophores CLR1501 (green with excitation/emission peaks 500/517 nm) and CLR1502 (NIR with excitation/emission peaks 760/778 nm) were reported to have higher tumor-to-normal brain fluorescence than 5-ALA (7.23 ± 1.63 and 9.28 ± 1.08 vs. 4.81 ± 0.92, respectively) in a mouse xenograft glioblastoma model (
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 (
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 (
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 (
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; Recombinant antibody mimicking binders: Affibodies: small (6.5-kDa) single domain engineered proteins that bind target proteins, imitating antibodies ( Nanobodies: a single variable domain of an antibody, which is capable of specific binding ( Aptamers: short single strands of nucleic acids, which are capable of specific binding (
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 (
The major drawbacks of targeted molecular probes are uneven passive distribution and non-specific binding. Dual-labeled probes were designed to address this limitation (
In a 2014 report, Ghosh et al. described a novel targeted probe construct containing a single-walled carbon nanotube as a fluorescent tag (
A targeted probe consisting of fluorescent gold nanoparticles conjugated with diatrizoic acid and AS1411 aptamer (
Another agent, BLZ-100, a tumor ligand chlorotoxin conjugated to ICG, was shown to have high affinity to human gliomas in mice (
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 (
IRDye800CW-labeled anti-EGFR nanobody 7D12 was compared to the full antibody cetuximab and showed better penetration and distribution of the nanobody probe
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
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.
Several different technologies are applied in fluorescence-guided resection of brain tumors. These technologies are classified into several categories ( Wide-field fluorescence imaging: Commercial operative microscopes with built-in fluorescence channels; Custom modified surgical microscopes; Surgical endoscopes equipped with fluorescence modules; Non-microscope fluorescent excitation systems with emission detecting devices. Quantitative fluorescence systems: Spectroscopic tools for imaging one region at a time; Laboratory grade stand-alone systems; Combination systems that integrate fluorescence with spatial imaging. Intraoperative high-resolution endomicroscopy.
Wide-field fluorescence imaging refers to non-microscopic, endoscopic, or microsurgery in which full fields of view are seen continuously through the eyepieces or on the screen during image acquisition at a rapid frame rate with a digital detector array (CMOS or CCD cameras) (
The use of custom operative microscopes with modules to measure fluorescein (
Fluorophores that emit in the NIR spectrum require CCD cameras or other detection technologies. An ICG (NIR) module does not require an operative microscope
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 (
A new endoscopic technology, scanning fiber endoscopy (
A concept for a low-cost fluorescein detection system for glioma surgery (
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 (
Absorption, scatter, anisotropy, and autofluorescence of the tumor and background tissue play important roles in the detection of the fluorophore signal, especially at low signal levels. Thus corrections for the optical properties of the tissues provide qualitative information about the fluorescence intensity in the area of interest (
A spectrally resolved quantitative fluorescence imaging system with submillimeter spatial resolution (214–125 μm) has been integrated with a conventional operative microscope (
The rationale for high-resolution intraoperative imaging is the inherent limitations of wide-field fluorescence microscopy and the desire for precise tissue visualization at the cellular level. Fiber optic confocal microscopy was invented in 1988 (
Confocal endomicroscopy may also be employed as a rapid diagnostic tool for biopsy specimens in
Although 5-ALA visualization was not optimal due to the limitation of the probe excitation profile, the other fluorescent stains clearly showed the histological features of the tumor cells and margins in a murine brain tumor model. Normal morphology in various brain regions was also clearly discernible in a large animal model (pig) using confocal endomicroscopy with topical acridine orange. Selective detection of ICG in a murine glioblastoma model was also shown using a clinical-grade, NIR confocal endomicroscopic system (
The initial experience with the Cellvizio confocal endomicroscope for immediate
The inherent limitations of the intraoperative confocal endomicroscope are a narrow field of view, the image appearing on a separate display, and the necessity of non-standard image analysis and interpretation, along with limited resolution, laser excitation spectrum, and corresponding detection power. Several computer image processing methods have been proposed to improve the diagnostic value of these small-field-of-view systems. For example, an image stitching technique has been applied to create panoramic wide-field images (
Approval of targeted fluorescent probes for clinical use will likely stimulate the refinement of confocal endomicroscopy and its broad clinical use in neurosurgery and tumor pathology. These two technologies are complimentary and allow tailored, tumor-specific resections for personalized patient treatment and, certainly, precision tumor surgery. The ability to interrogate the tumor border optically is of significant advantage in the acquisition of selective biopsies of higher diagnostic yield. Such a situation could improve the neurosurgery–neuropathology workflow for increased efficiency.
Several studies in optics, bioengineering, biotechnology, experimental oncology, and biochemistry have advanced the field of fluorescence-guided surgery in the preclinical arena (
Pulsed-light imaging is a technology that exploits pulsed excitation light and time-gated detection. It allows fluorescence imaging under normal operating room light conditions with high detection sensitivity (
A novel type of fluorophore, quantum dots, appears to be a relevant nanotechnology for fluorescence. The quantum dot is a 5- to 20-nm nanocrystal made from a semiconductor material that acts like a traditional fluorophore but works by a different mechanism. The emitted wavelength of the quantum dot depends on the size of the crystal. Fluorescent probes with the desired emission band may be designed. The main advantages of the quantum dot are much longer excitation life leading to photostability. The color of the emitted light may be tuned to the size of the probe. However, the safety of quantum dots is significant because larger quantum dots may not be well cleared, and the long-term effects of accumulation are unknown (
Another important parameter of future fluorophore probes is the size of the molecule, in which small targeted molecules, even with lower affinity, show better delineation of tumor boundaries most likely due to crossing the BBB more easily (
Some other emerging technologies may help in differentiating normal tissue from brain tumor tissue. For example, optical coherence tomography does not require any targeting agent. The technology utilizes differences in the optical signatures of the tissues to differentiate brain tumor from normal tissue, as shown in an animal study (
Intraoperative fluorescence imaging is capable of maximizing tumor tissue resection, providing rapid histopathological diagnoses based on innovative fluorophore probes and tools for intraoperative visualization. What is clear is that we sit on the threshold of technology that will enable neurosurgeons to see tumor cells in groups or individually in real time, which will allow tailoring or personalization of neurosurgery in terms of tumor resection. The term “theranostics” was coined to define ongoing efforts to develop precise, specific, individualized diagnostics and therapeutics for various diseases. For neurosurgery, we are adapting true precision modalities or biomarker techniques into diagnosis, including the imaging techniques described here, and this facilitates precise approaches to surgery. Cell-specific visualization will make possible the optimal surgical treatment of invading tumors such as gliomas that are composed of heterogeneous tissue with various genetic and metabolic characteristics. Therefore, the previously impossible may become routinely possible. If invading tumor cells are discovered in eloquent cortex, which is not normally resected, the neurosurgeon might be able to proceed on a cell-by-cell basis, targeting only tumor cells. Improved imaging technologies will bring about novel techniques to target or remove tissue or even individual cells. The advantages of such techniques are better surgical outcomes as nearly “cell-by-cell” or precision surgery becomes possible. Such surgical advancements will undoubtedly come with additional responsibilities, decisions, and challenges to be faced by both the neurosurgeon and patient.
All authors made substantial contributions to the conception or design of the work.
This research is supported in part by Zeiss, but they did not take part in the design of the experiments, examination of data, or writing of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors are grateful to the Neuroscience Publications staff at Barrow Neurological Institute for editing support.
This research was supported by funds from the Barrow Neurological Foundation, the Women’s Board of the Barrow Neurological Institute, and the Newsome Family Endowment in Neurosurgery to Dr. Mark Preul and by the Russian Science Foundation (Project 14-32-0006).
5-ALA, 5-aminolevulenic acid; BBB, blood–brain barrier; EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; GTR, gross total resection; ICG, indocyanine green; NIR, near-infrared; PDT, photodynamic therapy; PpIX, protoporphyrin IX; ROS, reactive oxygen species.
1
2