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In vivo wide-area cellular imaging by side-view endomicroscopy
2010 Nature America, Inc. All rights reserved.
Pilhan Kim1, Euiheon Chung2, Hiroshi Yamashita2, Kenneth E Hung3, Atsushi Mizoguchi4,
Raju Kucherlapati5, Dai Fukumura2, Rakesh K Jain2 & Seok H Yun1,6,7
In vivo imaging of small animals offers several possibilitiesfor studying normal and disease biology, but visualizing organs with single-cell resolution is challenging. We describe rotational side-view confocal endomicroscopy, which enables cellular imaging of gastrointestinal and respiratory tracts in mice and may be extensible to imaging organ parenchyma such as cerebral cortex. We monitored cell inltration, vascular changes and tumor progression during inammation and tumorigenesis in colon over several months.
The gastrointestinal tract and respiratory airways are majorsitesofimmunologicalchallenge.Theinterplayamongmicro-organisms,theepithelialbarrier,immunityandgeneticsiscriticaltocontrolorganismhomeostasis.Impairmentofoneorsomeofthesefactorscanleadtohomeostaticimbalance,causingdiseasesuchasinflammatoryboweldiseases1,dietproblems,infectiouslungdiseases2andcancer.Toinvestigatethecomplexmucosalimmunesystemanddiseasesrelatedtoit,smallanimalmodels,particularlymice,havebeenwidelyused.Animalstudieshaveprimarilyreliedonhistologicalexaminationsofexcisedtissuesex vivo. Although well established, this approach providesonlystaticinformationataspecifictimepointandthereforeis inadequate for investigating dynamic longitudinal eventsinvolved,forexample,inhost-microbialinteractions,immunereactionsandtumordevelopment.Real-timeintravitalfluorescencemicroscopycouldbeapowerfultechniqueforvisualizingsuchprocessesinnaturalenvironments3,4.Untilnow,however,in vivocellularimagingofthemucosainsmallanimalshasbeendifficultowingtothelackofanoninvasiveendoscopicmethodwithhighresolutionandeasymaneuverability.
Recently,considerableefforthasbeenmadetorealizehigh-resolution,minimallyinvasiveendoscopyinmice.Laser-scanning
1WellmanCenterforPhotomedicine,DepartmentofDermatologyand2EdwinL.SteeleLabforTumorBiology,DepartmentofRadiationOncology,HarvardMedicalSchoolandMassachusettsGeneralHospital,Boston,Massachusetts,USA.3DivisionofGastroenterology,TuftsMedicalCenter,Boston,Massachusetts,USA.4DepartmentofPathology,CenterfortheStudyofInflammatoryBowelDisease,HarvardMedicalSchoolandMassachusettsGeneralHospital,Boston,Massachusetts,USA.
5DepartmentofGenetics,BrighamandWomensHospital,Boston,Massachusetts,USA.6GraduateSchoolofNanoscienceandTechnology,KoreaAdvancedInstituteofScienceandTechnology,Yuseong-gu,Daejeon,Korea.7TheHarvardMassachusettsInstituteofTechnologyDivisionofHealthSciencesandTechnology,Cambridge,Massachusetts,USA.CorrespondenceshouldbeaddressedtoS.H.Y.([email protected]).
RECEIVED 19 AUGUST 2009; ACCEPTED 7 JANUARY 2010; PUBLISHED ONLINE 14 MARCH 2010; http://www.nature.com/doifinder/10.1038/nmeth.1440
Web End =DOI:10.1038/NMETH.1440
confocalendomicroscopy,basedonaresonantlyvibratingfiberorafiberbundle,hasshownapotentialforcellularexaminationofthecolon5,6.Thefront-viewconfigurationoftheinstrumentsrequiresdirectcontactoftheprobeperpendiculartotheintestinalwall.Althoughsuchacontactprobewouldbeviableinahumanindividual5,ithasprovenverydifficulttomaneuverinsmallanimals,suchasmice,becauseoftheirsmalllumendiameters.Noncontactendoscopeshavebeendevelopedtoprovideafish-eyeviewsimilartoconventionalclinicalcolonoscopy7,8.Butthisapproachrequires
alargedepthoffieldforagivenlimitedaperturesize,andtherefore,microscopicresolutioncouldnotbeachieved.Microendoscopyusing graded-index (GRIN) lenses has been demonstrated forimagingbrainneuralcircuitry9andmusclekinetics10inmice.However,thefieldofviewofsuchahigh-resolutionprobeistypicallyonly5%ofitscross-sectionalarea,seriouslylimitingthesizeoftissueinterrogatedatagiveninsertionsite.
Herewedescribeanewapproachbasedonaside-viewmicro-probethatovercomesthelimitationsofcurrentendoscopesandenableswide-areacellular-levelfluorescenceimagingoftissueinlivemice.Contactbetweentheviewwindowandluminalwallmakesiteasytonavigatealongthetractbyrotationandtranslationoftheprobe.Thisallowedustoobtainacomprehensivemapoffluorescentlylabeledcellsandmicrovasculatureinthemucosain vivoatmultipletimepoints.Wedemonstratethenewpossibilitiesenabledbythistechnologyinmousemodelsofcolitisandcolorectaltumor.
Tofabricateahigh-resolutionside-viewendoscopicprobe,wemodifieda1-mm-diametertripletGRINlensmicroendoscope9,11
andattachedanaluminum-coatedright-angleprismatthedistalend(Fig. 1a).Wethensealedtheopticalunitinastainlessprotectionsleevewithatransparentepoxy.Theassembledrigidendoscopehasanouterdiameterof1.25mmandalengthof50mm.Weintegratedtheendoscopeintoacustom-builtvideo-ratescanning-laserconfocalmicroscope11.Thelaserbeamisrasterscannedoverafixedx-yplaneattheproximalend,soweusedasimpleimagerotationtoconvertthex-ydatatoacircumference-lumenframe(Fig. 1b).
Theendoscopehadafieldofviewof250m250mandtrans-verseandaxialresolutionofabout1and10m,respectively,intheair.Theendoscopecouldberotatedendlesslytochangetheimagingplanealongthecircumference.Tochangetheviewplanealongthelumen,wemovedthemouseaxiallybyusingamotorizedtranslationstage.Thefocaldepth(z)inthetissuewascontrolledexternallywithouthavingtomovetheendoscopeormouse,simplybytranslatingthecoupling40objectivelenstochangethedistancetotheendoscope(Fig. 1candSupplementary Fig. 1).
NATURE METHODS | VOL.7 NO.4 | APRIL2010 | 303
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Figure 1 | In vivo side-view endomicroscopy.(a) Schematic of a laser-scanning side-viewendoscope. The raster-scanned beam in x and ydimensions is relayed by grade-index lenses in the probe and directed by a 90 prism to aside-view window. and represent the axialand circumferential coordinates, respectively, in the imaging plane. denotes the rotation angle of the probe, or the angle between x and axes.(b) Coordinate transform between the proximal(x-y) and distal (-) imaging planes. The traceof the raster-scanned beam is depicted in bluesolid lines. (c) In the imaging setup, the laserbeam emitted from the endoscope is projectedto a stage. Dashed lines depict the outline ofthe beam diverging after going through theimaging plane. Inset, distal tip of the probe. (d) Three-dimensional rendered fluorescenceimage of the vasculature in the descending colon of a normal C57B6/L mouse....