Intraoperative Fluorescence Imaging For Personalized Brain .
Reviewpublished: 17 October 2016doi: 10.3389/fsurg.2016.00055IEvgenii Belykh1,2,3,4, Nikolay L. Martirosyan1,2, Kaan Yagmurlu1, Eric J. Miller5,Jennifer M. Eschbacher1, Mohammadhassan Izadyyazdanabadi1,2,Liudmila A. Bardonova1,3,4, Vadim A. Byvaltsev3,4, Peter Nakaji1 and Mark C. Preul1*Department of Neurosurgery, St. Joseph’s Hospital and Medical Center, Barrow Neurological Institute, Phoenix, AZ, USA,School of Life Sciences, Arizona State University, Tempe, AZ, USA, 3 Laboratory of Neurosurgery, Irkutsk Scientific Center ofSurgery and Traumatology, Irkutsk, Russia, 4 Irkutsk State Medical University, Irkutsk, Russia, 5 University of Arizona College ofMedicine – Phoenix, Phoenix, AZ, USA12IEdited by:Eberval Figueiredo,Universidade de São Paulo, BrazilReviewed by:Yasunori Fujimoto,Osaka University, JapanAndrei Fernandes Joaquim,University of Campinas, Brazil*Correspondence:Mark C. Preulneuropub@dignityhealth.orgSpecialty section:This article was submittedto Neurosurgery,a section of the journalFrontiers in SurgeryReceived: 27 June 2016Accepted: 12 September 2016Published: 17 October 2016Citation:Belykh E, Martirosyan NL,Yagmurlu K, Miller EJ,Eschbacher JM,Izadyyazdanabadi M, Bardonova LA,Byvaltsev VA, Nakaji P and Preul MC(2016) Intraoperative FluorescenceImaging for Personalized BrainTumor Resection: Current Stateand Future Directions.Front. Surg. 3:55.doi: 10.3389/fsurg.2016.00055Frontiers in Surgery www.frontiersin.orgKeywords: 5-ALA, confocal, endomicroscopy, fluorescein, fluorescence-guided surgery, fluorescent probe,glioma, ICGAbbreviations: 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, photodynamictherapy; PpIX, protoporphyrin IX; ROS, reactive oxygen species.1October 2016 Volume 3 Article 55
Belykh et al.Fluorescence-Guided Precision Neuro-OncologyINTRODUCTIONsodium was in use even earlier than the first operative microscopesused by neurosurgeons and was pioneered by Kurze in 1957 (24).However, fluorescent dye technology did not gain widespreadacceptance due to the high rate of background fluorescencefrom normal brain tissue and the shortcomings of visualizationtechnologies (25). Fluorescein injection for cerebrovascular andtumor surgery was studied in detail by Feindel in the 1960s (26).A major step after the introduction of CT with contrast injection(23) was gadolinium-enhanced MRI, introduced around 1987,that allowed even more precise tumor mass visualization and precise anatomical co-registration for planning surgery (27). Infraredframeless neuronavigation systems resulting from developmentsin stereotactic and computer technologies were rapidly adoptedin neurosurgical operating rooms in the late 1980s (28–30). Thevirtual linkage of neuroimaging and intraoperative anatomyallowed a precision of nearly 2 mm, selection of the best approachtrajectory (31), and radically improved the surgeon’s intraoperative orientation. The main drawback of neuronavigation remainsbrain shift [1 cm on average (32)] as a consequence of openingthe cranium, which significantly limits the accuracy of determining an infiltrative tumor border (33). Despite software advances(32), intraoperative ultrasound (34, 35), and intraoperative MRIcorrections (36), the current (2015) technologies do not providethe desired accuracy for consistent, precise, and extensive resection (37). The main drawback continues to be that MR and CTimage characteristics are not directly indicative of regional tissuetype and cannot provide clinically applicable imaging at or nearcellular resolution.Accurate visualization of brain tumors marked by fluorescentprobes and even residual tumor cells is possible with emergingnew technologies. These emerging technologies are expectedto become state-of-the-art tools to maximize customized braintumor treatment. These technologies are the logical extension ofthe evolution of the search for precision in brain tumor surgery.Such technologies will allow real-time imaging interrogation ofthe brain during surgery at the cellular resolution to maximize ortailor brain tumor resection.This review summarizes recent achievements and futureperspectives of clinical, laboratory, and translational studies thatbring fluorescence-guided neurosurgery to the cellular level,thereby allowing for individualized brain tumor resections,representing a crucial breakthrough in this field.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 currenttreatment 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 survivalof patients with glioblastomas (1, 2), low-grade gliomas (3),meningiomas (4), and other intracranial malignancies. Therefore,the initial treatment goal should be the maximal removal of thetumor mass. Tumor mass resection is guided intraoperativelyby anatomically registered images (usually CT and MRI)incorporated into a stereotactically based image-guided surgeryplatform. Such a surgical strategy becomes a balance of aggressivetumor removal while producing no new or further permanentneurological deficit. Although there are characteristics of imagesfrom CT and MRI that indicate what tumor, necrosis, or edematous cortex is, the main focus of surgery is achieving maximalresection of the invading tumor front. In light of this, researchershave endeavored to make any invisible part of the tumor visibleusing advanced imaging techniques.Advances in imaging began with the philosophies of cerebral localization and function, while techniques for improvingprecision and the customization of brain tumor surgery can betraced to the late nineteenth century. The evolution of imagingtechniques in neurosurgery began with the first attempts at craniometric localization of intracranial lesions (5). The introduction of X-rays in neurosurgery in 1896 by Krause and Cushing(6), pneumoencephalography in 1919 by Dandy (7, 8), andcerebral angiography specifically for brain tumors by Moniz in1927 (9, 10) were the first steps in preoperative imaging diagnosisof brain tumors, which was previously possible only by clinicalneurological examination. Intraoperative stimulation in awakepatients to increase the safety of tumor resection was performedby Thomas and Cushing (11). This stimulation was possible dueto Cushing’s previous experience in mapping the motor cortexof primates in 1902 in the physiology laboratory of Sherrington(12). However, the origins of intraoperative neurophysiologyfor functional localization have roots in the works of Betz (13),Ferrier (14), Fritsch and Hitzig (15), and Clark and Horsley (16).Since the beginning of the twentieth century, several neurosurgeons, most notably Penfield in 1928, have used intraoperativebrain stimulation extensively to map the cortex to guide braintumor resection and surgical treatment of epilepsy (17). Buildingon earlier work, intraoperative electrophysiological monitoringand cortical and subcortical mapping performed with the patientconscious remain state-of-the-art methods to elicit functions ofbrain areas and define and personalize safe boundaries of tumorresection (18, 19).Techniques for visually identifying the tumor mass began inthe mid-twentieth century. The application of the fluorescent dyefluorescein sodium to highlight tumor tissue during its removalwas introduced in neurosurgery by Moore et al. in 1948 (20),decades before computed tomographic (CT) scanning was introduced into broad clinical practice (1973) (21–23). FluoresceinFrontiers in Surgery www.frontiersin.orgFLUORESCENT DYES INNEUROSURGERYIn the last decade (2006–2016), the number of fluorescentstains and cellular tags used in preclinical studies has increasedsignificantly, with many novel fluorophores awaiting approval forclinical use. The fluorescent probes and dyes discussed in thisreview are summarized in Table 1 (25, 38–74). Three fluorescent contrast agents that have been studied and used in humanneurosurgical procedures are fluorescein sodium, indocyaninegreen (ICG), and 5-aminolevulinic acid (5-ALA), althoughnot all are approved by regulatory committees in all countries.Other fluorophores (including acridine orange, acriflavine, cresyl2October 2016 Volume 3 Article 55
Name of probeReportedreadingemissionwavelengthUsed equipmentSpecies testedAdvantages675 and 745 nm(in vivo)800 nm(in vivo)1. IVIS Spectrum (PerkinElmer, Inc.)2. Multispectral Fluorescence CameraSystem (Institute for Biological andMedical Imaging, Technical University,Munich, Germany and SurgOptix Inc.,Redwood Shores, CA, USA), in vivo3. Olympus Fluoview 300 Confocal ScanBox mounted on an Olympus IX 71inverted microscope (Olympus AmericaInc.), ex vivo4. Pearl Imaging System(LI-COR Biosciences) in vivoXenograft mice model(human ovarian, breast,and gastric cancers)Long half-life forDistinguish submillimeter lesionsdetecting tumors.intraoperatively. Longer lastingLong elimination timeand more accurate signal forVEGF and EGFR2 than ICG alone.Bevacizumab-800CW fluorescencedetection in extracellular matrix,trastuzumab-800CW fluorescencedetection on tumor cell surfaceIV, 6 days(optimal time)IRDye 800CW-labeledhuman EGFR 2[Trastuzumab (38);Erbitux (39)]675 and 745 nm(in vivo); 685 and785 nm800 nm(in vivo); 720and 820 nm1. IVIS Spectrum (PerkinElmer, Inc.)2. Multispectral Fluorescence CameraSystem (Institute for Biological andMedical Imaging, Technical Universityand SurgOptix Inc.), in vivo3. Olympus Fluoview 300 Confocal ScanBox mounted on an Olympus IX 71inverted microscope (Olympus AmericaInc.), ex vivo4. Pearl Imaging System (LI-CORBiosciences) in vivoXenograft mice model(human ovarian, breast,and gastric cancers);Xenograft mice model(human breast cancerlymph metastasis)Long half-life forDistinguish submillimeter lesionsdetecting tumors.intraoperatively. Longer lastingLong elimination timeand more accurate signal forVEGF and EGFR2 than ICG alone.Bevacizumab-800CW fluorescencedetection in extracellular matrix,trastuzumab-800CW fluorescencedetection on tumor cell surfaceIV, 3–6 days(optimaltime); 3 h forlymph nodevisualizationIRDye 800CW-labeledanti-EGFR nanobody7D12 (40, 41)760 nm;656–678 nm;745–779 nm774 nm;700 nm;800 nm1. IVIS Lumina System (PerkinElmer, Inc.)with ICG filter sets2. FLARE imaging system (Beth IsraelDeaconess Medical Center)3. IVIS Spectrum (PerkinElmer, Inc.)Xenograft mice (humanepidermoid carcinoma);xenograft mice (humanmetastatic oral squamouscell carcinoma)Better tumor penetration anddistribution of nanobody probein vivo (vs. cetuximab full antibody).Significantly higher tumor tobackground fluorescence (vs.cetuximab full antibody)Not mentionedIV, 30 min(earliest); 2 h(optimal); or 24 h(optimal)IRDye 680RD labeledEGFR inhibitor(cetuximab) (42)620 nm650–800 nmOdyssey Infrared Imaging System (LI-CORBiosciences, Lincoln, Nebraska), ex vivoXenograft mice (humanU251 glioma)Higher affinity for tumor than antiEGFR targeted affibody used insame studyConcentration ofantibody in tumorfocused primarily inthe centerIV, 1 hIRDye 800CW-labeledanti-EGFR targetedaffibody (42)720 nm730–900 nmOdyssey Infrared Imaging System (LI-CORBiosciences), ex vivoXenograft mice (humanU251 glioma)Smaller size molecule results inbetter penetration of BBB. Higherconcentration in outer tumor thanantibody30 times lower affinitythan antibody anda shorter plasmahalf-lifeIV, 1 hTargeted probesIRDye 800CW-labeledVEGF (38)(Bevacizumab)DisadvantagesMode ofadministrationand timeto imaging(unless notedotherwise)3October 2016 Volume 3 Article 55(Continued)Fluorescence-Guided Precision Neuro-OncologyReportedexcitationwavelengthBelykh et al.Frontiers in Surgery www.frontiersin.orgTABLE 1 Summary of published preclinical and early clinical data on probes and imaging equipment for potential personalized fluorescence-guided brain tumor surgery.
onwavelengthUsed equipmentIRDye 800CW-labeledchemokine stromalcell derived factor-1(SDF-1) (43)685 and 785 nm702 or789 nmIRDye 800CW-labeledanti-CD105monoclonal antibody(angiogenesis related)(44)778 nm806 nmCy5.5-labeled EGFRinhibitor (cetuximab)(45)683 nm (max);630–670 nm(range used inexperiment)707 nm (max); 1. Leica MZFL3 stereo research685–735 nmmicroscope (Leica Microsystems,(range used inBannockburn, IL, USA) fitted with aexperiment)GFP and Cy5.5 filter and an ORCAER charge-coupled device camera(Hamamatsu, Bridgewater, NJ, USA)2. eXplore Optix time-domain fluorescenceimaging system (ART/GE Healthcare,Princeton, NJ, USA)Alexa-680 labeledinsulin-like growthfactor 1 receptor(IGF1 R) (AVE-1642conjugated Alexa680) (46)575–605 nm645–850 nmFolate–fluoresceinisothiocyanate probe(for folate receptor)(47)495 nm520 nmSpecies testedAdvantagesDisadvantagesMode ofadministrationand timeto imaging(unless notedotherwise)Pearl Imaging System (LI-COR Biosciences), Xenograft mice (A764in vivohuman glioma, MCF-7human breast cancer)Detected as low as 500 cellsin vitro. Specific for tumor cells.Signal persisted for daysLabeled bonemarrow, transientnon-specific labelingduring first 24 h wasobserved in the liverand skullIV, 1-hvisualizationof tumors andbackgroundstructures;24–92 hbackgroundfluorescencediminished,tumors remainedclearly visiblePearl Imaging System (LI-COR Biosciences)in vivo and in vitroTumor could be visualized as earlyas 30 min post-injection; may beused in the clinic for imaging tumorangiogenesisCD105 expressionis observed only onactively proliferatingtumor endothelialcellsIV, 30 min (early);16 h (optimal)Cell cultures: UM-SCC-1, Can be used to detect tumorsFaDu, CAL 27, and AB12; in vivoxenograft mice model(human head and necksquamous cell carcinomacell lines SCC-1, FaDu,CAL 27); mice withmouse mesotheliomaEGFR expression didnot correlate with thefluorescent intensityIV, 48–72 h(optimal)1. Maestro Imaging System (CRI), in vivo2. Olympus Fluoview FV500 laser scanningconfocal system (Olympus America Inc.)Xenograft mice model(MCF-7 human breastcancer cells)Can detect the downregulationof IGF1R after treatment with amonoclonal antibodyFurther studiesrequired to determinethe amount ofbackgroundfluorescenceproduced by IGF1R1 day (earliest);2 days(clear imaging)Intraoperative Multispectral FluorescenceCamera System (Institute for Biological andMedical Imaging, Technical University)Humans with ovariancancerHigh specificity for labelingFR-alpha expressing cells. Realtime image-guided excision offluorescent tumor deposits of size 1 mm was feasibleFour patientsexperienced milddiscomfort in theupper abdominalregion after injectionImagingcompleted 2–8 hafter injectionMice with 4T1 mousebreast cancer; humanMCF-7 breast cancercells in cultures(Continued)Fluorescence-Guided Precision Neuro-OncologyOctober 2016 Volume 3 Article 55Name of probeBelykh et al.Frontiers in Surgery www.frontiersin.orgTABLE 1 Continued
onwavelengthUsed equipmentSpecies testedAdvantagesDisadvantagesMode ofadministrationand timeto imaging(unless notedotherwise)BODIPY FL-labeledPARP inhibitor(Olaparib) (48)503 nm515 nmMaestro Imaging System (CRI)Xenograft mice model(U87 MG and U251 MGhuman glioblastomas)High specificity for the DNA repairenzyme PARP1 with therapeuticeffect. Promising new targetedantitumor drug, which is alreadyin clinical trials. High tumorbackground fluorescent ratio.Toxicity profile is known and similarto OlaparibNot mentioned60–180 min(optimal)Liposomes withRGD peptide andthe neuropeptideSP, gadolinium,Indium-111,Rhodamine-B (49)554 nm576 nmZeiss LSM 510 Microscope (Carl ZeissMeditec AG, Jena, Germany)Cultured mouse fibroblastcells with U87 MG humanglioblastoma and M21human melanoma tumorcells (in vitro)Combination of radioactive,fluorescent, and magneticresonance imaging signaling;multifunctionality of liposomes as acarrier of different probesModerate tumoruptakeIn vitrofluorescencemicroscopy wascompleted aftertumor cells weregrown in mousefibroblast cultureZW800-1 zwitterionicNIR fluorophore (50,51)750 25 nm;773 nm810 20 nm;790 nm1. FLARE Imaging System (Beth IsraelDeaconess Medical Center)2. FLARE Imaging System (Beth IsraelDeaconess Medical Center)3. Pearl Small Animal Imaging System(LI-COR Biosciences)Xenograft mice model(M21 human melanoma,Lewis lung carcinoma,HT-29 human colorectaladenocarcinoma)Higher tumor-to-background ratiothan IRDye800-CW and Cy5.5Wash-out of dyefrom tumors startedoccurring at 4 h (dyestill present at 24 h)4 h, low visibilityat 4 h, highestvisibility from 24to 72 hM13-stabilizedsingle-walled carbonnanotubes(SBP-M13-SWNTs)(52)808 nm950–1400 nm Liquid nitrogen-cooled OMA V 2D InGaAsarray detector with a 256 320 pixel array(Princeton Instruments) coupled with SWIR25 NIR camera lens (Navitar, Rochester,NY, USA)Xenograft mice model(OVCAR8 human ovarianepithelial carcinoma)Stable and showed 10 times moreselective fluorescent staining ofovarian tumor cells than sameconstruct without targetingpeptide. Nanotube fluorescenceintensity relative to background(5.5 1.2) was superior to sameconstruct labeled with other NIRAlexaFluor750 dye (3.1 0.42) orFITC (0.96 0.10)Study did not assesspossible penetrationof the probe into thebrain24 hFluorescent goldnanoparticlesconjugated withdiatrizoic acid andAS1411 aptamer (53)400 nm620 nm (max)Xenograft micemodel (human lungadenocarcinoma)separate MCF-7 cellassaySpecific binding to tumor cellsdue to AS1411 aptamer, whichtargets nucleolin. Allowed X-rayvisualization due to high electrondensity of gold nanoparticlesSmall sample size(n 6)30 min1. Ultra-VIEW RS Confocal System(PerkinElmer, Inc., Waltham, MA, USA)2. IVIS (PerkinElmer, Inc.)(Continued)Fluorescence-Guided Precision Neuro-OncologyOctober 2016 Volume 3 Article 55Name of probeBelykh et al.Frontiers in Surgery www.frontiersin.orgTABLE 1 Continued
6Name of issionwavelengthUsed equipmentSpecies testedAdvantagesDisadvantagesMode ofadministrationand timeto imaging(unless notedotherwise)Lymphoma-specificfluorescent (Alex488)switchable TD05aptamer (54)489 nm505–535 nmZeiss 710 laser Scanning ConfocalMicroscope (Carl Zeiss Meditec AG)equipped with a 40 /1.2NA water emersionobjective (ex vivo)Xenograft rat model(U251 human gliomaand Ramos human CNSlymphoma)Probe could rapidly and specificallyidentify human B cell lymphoma inbiopsies. System would be usefulfor discriminating non-operativeCNS B-cell lymphoma frommalignant glioma rapidly afterbiopsyEx vivo only studyTotal antibodystaining timewas 24 h andaptamer stainingtime was 1 h(ex vivo)Chlorotoxin (CTX)conjugated to ICG(BLZ-100) (55)785 nmNear-infraredspectrumCustom imaging system: 16-mm VIS-NIRCompact Fixed Focal Length Lens (EdmundOptics, Barrington, NJ, USA) coupled785-nm StopLine single-notch filter, NF03–785E-25 (Semrock, Rochester, NY, USA)Xenograft micemodel (LN229 humanglioblastoma)High affinity to human gliomasNot mentioned48 hNot givenKodak In Vivo Imaging System F(Kodak, Rochester, NY, USA)Xenograft mice model(U87MG humanglioblastoma)Selective uptake. May haveadvantages over CTX-Cy5.5 probedue smaller sizeFluorescein labelingwas less than ideal,could be exchangedfor more intensefluorophore1h505–530 nm1. In-house-made portable fluorescencecamera for ex vivo tumor specimens2. Zeiss LSM510 Microscope (Carl ZeissMeditec AG)Human breast cancertissue samples; breastcancer cell cultureHigh sensitivity and spatialresolutionFast processing: evaluation canbe done within 5 min after probeapplicationIn breast cancer,this method cannotdistinguish malignantand benign regions5 min5-Carboxyfluorescein Blue light(FAM)-labeledfluorescent probeconsisting of tLyP-1small peptide targetedto the neuropilinreceptors(FAM-tLyP-1) (56)Activity-based probesModified488 nmhydroxymethylrhodamine green(gGlu-HMRG) (57)749 nm775 nmSurgical Navigation System (Institute ofAutomation, Chinese Academy of Sciences,Beijing, China) (59)Mice with 4T1-luc breastcancer tumorsImaging method offered precisedetection of the orthotopicbreast tumors and metastasesintraoperatively in real timeNot mentionedIV, 6 h(fluorescentsignal observed);24–36 h gregationfluorescent probe(C-SNAF) (60)635 25 nm670–900 nmMaestro Hyperspectral Fluorescent ImagingSystem (CRI)Xenograft mice model(subcutaneous HeLatumors)Highly feasible for imaging ofdrug-induced tumor apoptosisin vivo, signal strengthens as tumorcells dieNot mentionedIV, 1 h(Continued)Fluorescence-Guided Precision Neuro-OncologyOctober 2016 Volume 3 Article 55MMPSense 750 FAST(MMP-750) (58)Belykh et al.Frontiers in Surgery www.frontiersin.orgTABLE 1 Continued
Name of probe7ReportedreadingemissionwavelengthUsed equipmentSpecies tested647 nm675–725 nm1. Olympus IX70 confocal microscope(Olympus America, Inc.)2. Ultra-VIEW Confocal Laser ScanningMicroscope (PerkinElmer, Inc.)Produced visible color change inCell cultures: 9L rattumor cell linesgliosarcoma,MDA-MB-435 humanmelanoma, MCF-7 humanbreast cancerIron oxide magneticNH2-CLIOnanoparticles labeledwith Cy5.5(Cy5.5-CLIO) (62)Not givenNot given1. Custom-built surface reflectance imaging Rat 9L gliosarcoma tumormodelsystem (Siemens Medical Systems,Erlangen, Germany), in vivo2. Zeiss LSM 5 Pascal (Carl Zeiss MeditecAG, Jena, Germany), ex vivoCyto647 labeledanti-EGFRantibody-conjugatedSERS-taggedgold nanoparticles(antibodyPanitumumab) (63)642 nm(Olympus);785 nm (Raman)700–775 nm1. Olympus IX81 inverted fluorescencemicroscope (Olympus America, Inc.)2. Hamamatsu Back-Thinned EM-CCDcamera, 9100-13 (Hamamatsu,Bridgewater, NJ, USA)3. Spinning Disk Confocal ScanningRaman Microscope (Renishaw, Wottonunder-Edge, UK)In vitro cell cultures:rat gliosarcoma cellline 9L; rat C6 glioma;human GBM cells U87,A172, U251, U373;normal fetal humanastrocytes; primaryoligodendroglioma tumorcells BT2012036; andGBM adherent stem cellline GLINS1400–410 nmviolet620–720 nmred1. VWCE2. Zeiss Pentero Microscope (Carl ZeissSurgical GmbH)780 nm 795 nm1. VWCE2. Zeiss Pentero Microscope (Carl ZeissSurgical, GmbH)3. Zeiss LSM710 (Carl Zeiss Surgical,GmbH)NanoparticlesPolyacrylamidebased nanoparticlesloaded with ICG orCoomassie blue dye(61)Others5-ALA thatmetabolically convertsinto fluorescent PpIXOctober 2016 Volume 3 Article 55Indocyanine green(ICG)DisadvantagesMode ofadministrationand timeto imaging(unless notedotherwise)Significant nonspecific binding wasobservedImaged after 2 hof incubationClear tumor border demarcationCo-localization based on MRIimagingElimination more predictable thanother nanoprobesNot as accurate astarget probes forin vivo tumor cellvisualizationIV, 24 hSelective uptake by tumorcells; unlike other fluorescentdyes, SERS nanoparticles haveenhanced photostabilityNot mentionedNot applicableStudies in human (25) and Studies have shown increasedin animals (64)extent of tumor resection with PpXIguided surgery; useful for braintumor biopsyDisruption ofBBB necessaryfor fluorophoreaccumulation (candecrease/varycontrast)Low fluorescenceintensity in low-gradegliomasOral, IV, 2 h (65)Mice with GL261 mouseglioma (66)ICG visualization canonly be displayed ona monitorIV, 15 minHuman (67)AdvantagesExtensively studied; hand-heldconfocal endomicroscope andLSM showed ICG selectivelystained glioma cells in mousemodel (66)Intraoperative administration at endof 5-ALA guided resection mayshow additional tumor tissue (67)(Continued)Fluorescence-Guided Precision Neuro-OncologyReportedexcitationwavelengthBelykh et al.Frontiers in Surgery www.frontiersin.orgTABLE 1 Continued
ReportedreadingemissionwavelengthUsed equipmentSpecies testedAdvantagesDisadvantagesMode ofadministrationand timeto imaging(unless notedotherwise)Fluorescein sodium(68)494 nm521 nm1. VWCE2. Zeiss Pentero Microscope (Carl ZeissSurgical GmbH)3. LSM710 (Carl Zeiss Surgical, GmbH)HumanConvenience for surgeon,surrounding tissue has morenatural colorRapidphotobleaching, nonspecific accumulationof fluorescein alongthe margins ofresection. Possibleextravasation alongwith edemaIV, 5 min (65)CLR1501 (69)500 nm517 nmNikon A1RSi Confocal Microscope (Nikon,Minato, Tokyo, Japan); IVIS Spectrumsystem (PerkinElmer, Inc.)Xenograft mousemodel (U251 humanglioblastoma, 22T,22CSC, 33CSC,105CSC patient derivedglioblastoma)Tumor-to-brain fluorescence ratiosimilar to 5-ALATumor must bevisualized onseparate monitorIV, 4 daysCLR1502 (69)760 nm778 nm1. IVIS Spectrum system (PerkinElmer, Inc.)2. Fluobeam 800(Fluoptics, Grenoble, France)3. Leica OH4 intraoperative microscopewith FL800 attachment (LeicaMicrosystems, Bannockburn, IL, USA)Xenograft mousemodel (U251 humanglioblastoma, 22T,22CSC, 33CSC,105CSC patient derivedglioblastoma)Tumor-to-brain fluorescence ratiosuperior to 5-ALATumor must bevisualized onseparate monitorIV, 4 daysCH1055 (70) 750 nm1055 nmIn-house-built NIR spectroscopy instrument Xenograft mice model(U87MG humanwith Acton SP2300i spectrometerglioblastoma)(Princeton Instruments, Trenton, NJ, USA)and Princeton OMA-V liquid-nitrogen-cooledInGaAs linear array detector(Princeton Instruments)High tumor-to-background signalTumor must beratiovisualized onPossibility for precise image-guided separate monitortumor removal in the model90% excreted through the kidneyswithin 24 hIV, 6 h (tumor isclearly visible);72 h (optimal)Acridine orange (64)488 nm505–700 nm(LSM);505–585(VWCE)1. VWCE2. Zeiss Pentero Microscope(Carl Zeiss Surgical GmbH)3. LSM710 (Carl Zeiss GmbH)Mice with GL261 glioma;swine normal brainSuitable for rapid intraoperativeex vivo analysis of glioma tissueCannot be usedin the brain due totoxicity profileTopicalapplication,immediatelyAcriflavine (64)405 nm (LSM);488 (VWCE)505–585 nm1. VWCE2. Zeiss Pentero Microscope(Carl Zeiss Surgical GmbH)3. LSM710 (Carl Zeiss GmbH)Mice with GL261 gliomaSuitable for rapid intraoperativeex vivo analysis of glioma tissueCannot be usedin the brain due totoxicity profileTopicalapplication,immediatelyCresyl violet (64)561 nm (LSM);488 nm (VWCE)620–655 nm(LSM);505–585 nm(VWCE)1. VWCE2. Zeiss Pentero Microscope(Carl Zeiss Surgical GmbH)3. LSM710 (Carl Zeiss GmbH)Mice with GL261 gliomaHighlights tumor boundaries ex vivo No current in vivobrain applicationLow signal-to-noiseratioOctober 2016 Volume 3 Article 55Topicalapplication,10 min(Continued)Fluorescence-Guided Precision Neuro-OncologyReportedexcitationwavelength8Name of probeBelykh et al.Frontiers in Surgery www.frontiersin.orgTABLE 1 Continued
585–615 nm(LSM);505–750 nm(VWCE) 520 nm 690 nm561 nm (LSM);488 nm (VWCE)402 nm642 nmSulforhodamine101SR101 (64)Demeclocycline (72)Methylene blue (74)Frontiers in Surgery www.frontiersin.orgviolet, and sulforhodamine 101) have been used for pulmonary,gastrointestinal, or gynecologic procedures and in ex vivo brainbiopsies. They have not been used directly in the human brain.Fluorescent probes and labels are classified based on the actualfluorescent molecule (i.e., intrinsic and extrinsic endogenousfluorophores) and excitation/emission profile and can be furthercategorized by their mechanism of action:BBB, blood–brain barrier; VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor; SERS, surface-enhanced Raman scattering, a nanoparticle tagging method to increase signal detection; FMI, fluorescencemolecular imaging; BCS, breast cancer surgery; LSM, laser scanning microscope; VWCE, visible wavelength confocal endomicroscope (Optiscan 5.1) (71).Highlights tumor cells ex vivoCustom confocal laser scanning microscope Human meningioma,glioma, andadenocarcinoma tissuesTopicalapplication,timing notreportedNon-specificHighlights tumor cells ex vivo andcorrelates with histology. Limiteddata suggest specificity for tumorcells (73)Custom confocal laser scanning microscope Human low- andhigh-grade glioma citationwavelengthNon-specificNon-specificStrongly labeled cells within thetumor and astrocytes within normalbrainXenograft rat model(U251 human glioma)1. VWCE2. Zeiss Pentero Microscope (Carl ZeissSurgical GmbH)3. LSM710 (Carl Zeiss GmbH)Topicalapplication,timing notreportedDisadvantagesAdvantagesSpecies testedUsed equipment1hFluorescence-Guided Precision Neuro-OncologyName of probeTABLE 1 ContinuedMode ofadministrationand timeto imaging(unless notedotherwise)Belykh et al.1. Passive fluorescent probes (ICG, fluorescein sodium, andother stains);2. Metabolic probes (5-ALA, activatable probes); and3. Targeted probes.One of the most important characteristics of the probes istheir 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 havea molecular weight more than 400–600 kDa (75). Based ontheir physical properties, photons with longer wavelengths inthe near-infrared (NIR) spectrum have greater tissue penetration and thus are advantageous for visualizing obscure residualtumor tissue or cells (Figure 1). However, fluorophore phototoxicity caused by the generation of reactive oxygen species (ROS)may be harmful to healthy cells. The principle of phototoxicityis also used in combination with fluorescence-guided tumorresection and photodynamic therapy (PDT). Most fluorescenceis associated with the production of some ROS and PDT effects(76). The combination of fluorescence-guided resection andpost-resection cavitary PDT with strong photosensitizers mayhave a synergistic effect and has already shown promising resultsin several clinical trials (77, 78). Nonetheless, this approach andthe exact methodology regarding the choice of a photosensitizer,excitation wavelengths, dosages, and other p
3. Olympus Fluoview 300 Confocal Scan Box mounted on an Olympus IX 71 inverted microscope (Olympus America Inc.), ex vivo 4. Pearl Imaging System (LI-COR Biosciences) in vivo Xenograft mice model (human ovarian, breast, and gastric cancers) Distinguish submillimeter lesions intr
Specialized Fluorescence Techniques 171 Single Molecule Fluorescence 172 Fluorescence Correlation Spectroscopy 173 Forster Resonance Energy Transfer 173 Imaging and Super-Resolution Imaging (Con-ventional and Lifetime) 174 Instrumentation and Laser Based Fluorescence Techniques 175 Nonlinear Emission Processes in Fluorescence Spectroscopy 176
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Practical fluorescence microscopy 37 4.1 Bright-field versus fluorescence microscopy 37 4.2 Epi-illumination fluorescence microscopy 37 4.3 Basic equipment and supplies for epi-illumination fluorescence . microscopy. This manual provides basic information on fluorescence microscopy
Introduction - Optical resolution - Optical sectioning with a laser scanning confocal microscope - Confocal fluorescence imaging Stimulated emission depletion (STED) microscopy Fluorescence resonance energy transfer (FRET) Fluorescence lifetime imaging Two photon excitation microscopy Conclusion @Physics, IIT Guwahati Page 3
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