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Poster WMIC Angiogenesis Cellvizio Dual Band
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Simultaneousimagingoftwofluorescentsignalsusinganewfiberedfluorescentconfocalmicroscopysystem
BertrandViellerobe1,IsabelleJanssens2,3,KarineGombert2,3,HediGharbi1,FrançoisLacombe1andFrédéricDucongé2,31)MaunaKeaTechnologies,9,rued’Enghien,75010Paris,France
2)CEA,I²BM,ServiceHospitalierFrédéricJoliot,4placedugénéralLeclerc,91401Orsay(France)3)INSERMU1023,UniversitéParisSud,Laboratoired’ImagerieMoléculaireExpérimentale,4placedugénéralLeclerc,91401Orsay(France)
Acknowledgments TheauthorswouldliketothankAnikitosGarofalakisforforhisvaluabletechnicalassistance for fDOT/CT imaging. This work was supported by grants from the“AgenceNa^onalepourlaRecherche”[projectsANR‐TechSANDo^magerandtheEuropean Molecular Imaging Laboratory (EMIL) network [EU contractLSH‐2004‐503569].
Introduction Today,confocalfluorescencemicroscopyandmul^photonmicroscopy are increasingly used for in vivo studies insmall animals. Such techniques allow studying thestructureandthephysiologyoflivingorganismatcellularscale. The major limita^ons of such imaging is that 1‐samplesneedtobeplacedconvenientlyonaconven^onalmicroscope stage which require extensive surgicalprepara^on, and 2‐ rapid image collec^on is required tominimize the effects of movement (such as animalbreathing). To solve this problem, novel confocalapproaches using fiber bundle‐based systems have beendeveloped by Mauna Kea Technologies (Paris, France).Such systems, named Cellvizio®, use extremely smallbundlesoffibers,0.3–2.6mmindiameterthatcancontainupwardsof30,000fibers.Eachfiberisusedforexcita^ondelivery and recovery of the emission back through thefibertoadetector.Hence,eachfibercanbecomparedasan independent insect eye. The absolute advantages ofthis apparatus are size, flexibility, and image collec^onspeed (up to of 12 frames/s). Up to now, two Cellvizio®systemswereavailableeitherwitha488nmora660nmlaser beam. Here, we describe the use of a new fiberbundle‐basedfluorescenceimagingprototype(Cellvizio®Dual Band) that can perform simultaneous excitaEonwith both lasers (488 nmand 660 nm) and recovery ofemission signal with two detectors. We validate thesystem comparing the biodistribu^on of a fluorescentRGD‐based probe (Angiostamp®) in different region of atumorxenogranaswellasindifferentorgansofamouse.Thisfluorescentprobeisknowntobindtheαvβ3Integrin,aproteinoverexpressedatthesurfaceofendothelialcellsduringangiogenesis[1].
Materials and methods ●EthicsStatementAll animal use procedures were in strict accordance with therecommenda^ons of the European Community (86/609/CEE) andtheFrenchNa^onalCommioee(décret87/848)forthecareanduseoflaboratoryanimals.●AnimalmodelFemale nudemice (~23 g) were subcutaneously injectedwith 106tumor cellsNIH‐MEN2A expressing the oncogen RETC634Y. Aner 15days,micehaveatumor(~30‐50mm3).●InvivofluorescenceimagingusingfDOT/CTAngiostamp (10 nmol) was intravenously injected into the tail ofanesthe^zedanimals.3Dfluorescence imageswereacquired3hor7hpost‐injec^onusingaprototypeop^calimager(TomoFluo3D).CTimaging was perform using the SkyScan 1178 high‐throughputmicro‐CT (Skyscan, Kon^ch, Belgium). Fusion of fDOTwith CTwasperformedusingtheBrainvisamedicalimagingprocessingsonware(hop://brainvisa.info/index_f.html)[2].●InvivofluorescenceimagingusingCellvizio®prototypeAner fDOT imaging , 1mg of FITC‐dextran (500 kDa) wasintravenously injected in animals before surgery. Then,Fluorescence imagingat the cellular levelwasperformedwith thefiberedconfocalmicroscopeCellvizio®DualBandfrom MaunaKeaTechnologies. The device consists in a flexible sub‐millimetricmicroprobe containing thousands of op^cal fibers that carry lightfrom two con^nuous laser source at 488 nm and 660 nm to theliving ^ssue. The fluorescence emioed aner excita^on by thefluorophores staining the ^ssue species is sent back to theapparatus,whereadedicatedsetofalgorithmsreconstructsimagesinreal^meataframerateof12framespersecond.Theprobethatwas used is a UltraMiniO probe with 30,000 op^cal fibers, a240x240µmfieldofview,anda1.4µmlateralresolu^on.
Results MacroscopicimagingofAngiostamp®usingfDOT/CTThe biodistribu^on of Angiostamp was first evaluatedusing fluorescence Diffuse Op^cal Tomography (fDOT) innude mouse bearing a subcutaneous xenogran tumorfromNIH/MEN2A cells. This imaging techniquehas beenconsiderably improvedsincepastdecadeandallowsnowreconstruc^ngandquan^fyingfluorescencesignalinthreedimensions insidesmallanimal. fDOT imaging fusedwithX‐Ray Computed Tomography (CT) demonstrated a highuptakeof the tracer in the tumor area. Interes^ngly, theuptake seems heterogeneous in the tumor and seemshigher in the booom of the tumor. In subcutaneousxenogranmodels, the tumour cannot easily grow to theskin where it cannot find a lot of nutrients, but itpreferen^ally invades the^ssuebelow.The tracer seemstohaveahigheruptakeinthatzonethatshouldberichinnewbloodvessels.However,althoughfDOTcannowdetectfluorescence inthenanomolarrange,ithassEllalow(afewmm)spaEalresoluEonthatcannotpermit tohaveaprecise ideaofthebiodistribuEonoftheprobeatthecellularscale.
Conclusions Usingtheendoscopicsystem,wedemonstratedthatwecansimultaneouslyobservethebiodistribu^onofAngiostamp®withbloodvessels.Weobservedahighaccumula^onofAngiostamp®suroundingbloodvesselsclose to tumor. Incontrast,noAngiostamp®waslocalisedclosetobloodvesselsofhealthy^ssuesuchasmuscle,spleen, liverorkidney.Hence,thenewCellvizio®allowsustoconfirmthatthemacroscopicimageobtainbyfDOTcorrespondstotumorangiogenesisimagingandmaybe also to uptake by tumor associated macrophages expressing theαvβ3 Integrin. In conclusion, the simultaneousmonitoring of two fluorescent signals by endomicroscopy can be useful to validate fluorescent probes used formacroscopicimaginganditopensanewavenuetomonitorinvivomoleculareventsatamicroscopicscale.
For further information Pleasecontact:[email protected]@cea.fr
Microscopic imaging of Angiostamp® using Cellvizio®DualBandFollowingfDOTimaging,themicewereinjectedwithFITC‐Dextran before imaging with the fiber bundle‐basedfluorescence imaging prototype (Cellvizio® Dual Band).Theinstrumentallowedtoacquiredinreal‐^meimageofblood vessels labeled with FITC‐Dextran and the signalfrom Angiostamp®. Thanks to the high flexibility of thesystemdifferentorganscaneasilybeenanalyzedaswellasdifferentpartofthetumorxenogran(scheme2).
Fig.1:BiodistribuEonofAngiostamp®analyzedbyfDOT/CTimagingFluorescence signal reconstructed in 3D (colored) was fused to CTimagingofthemouse(gray).
FITC-dextran AngioStamp ® Merge
Angiostamp®issurroundingthetumorbloodvessels
FITC-dextran AngioStamp ® Merge
Angiostamp®isnotsurroundingthebloodvesselsofmuscle
FITC-dextran AngioStamp ® Merge
FITC-dextran AngioStamp ® Merge
Angiostamp®isnotaccumulatedinliver
Angiostamp®isnotaccumulatedinspleen
FITC-dextran AngioStamp ® Merge
Angiostamp®iseliminatedbyglomerulusofkidney
FITC-dextran AngioStamp ® Merge
FITC-dextran AngioStamp ® Merge
Angiostamp®isnotsurroundingthebloodvesselsofmuscle
FITC-dextran AngioStamp ® Merge
FITC-dextran AngioStamp ® Merge
Angiostamp®isslightlyaccumulatedinliver
Angiostamp®isslightlyaccumulatedinspleen
FITC-dextran AngioStamp ® Merge
Angiostamp®iseliminatedbyglomerulusofkidney
FITC-dextran AngioStamp ® Merge
3h post-injection 7h post-injection
Merge FITC-dextran
Angiostamp®issurroundingthetumorbloodvessels
Literature cited [1] Garanger, E., Boturyn, D., Jin, Z., Dumy, P., Favrot, M.C. and Coll, J.L. (2005)
New multifunctional molecular conjugate vector for targeting, imaging, and therapy of tumors. Mol Ther, 12, 1168-1175.
[2] Garofalakis, A., Dubois, A., Kuhnast, B., Dupont, D.M., Janssens, I., Mackiewicz, N., Dolle, F., Tavitian, B. and Duconge, F. (2010) In vivo validation of free-space fluorescence tomography using nuclear imaging. Opt Lett, 35, 3024-3026.
Scheme2:IllustraEonofdifferentpartofthetumorthatcanbeimagedbytheCellvizio®DualBand
Scheme1:Cellvizio®DualBandsystem
