High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging.

0.41 MB·English

by  Pierce, Mark

JournalofVisualizedExperiments www.jove.com
VideoArticle
High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging
Mark Pierce1,Dihua Yu2,Rebecca Richards-Kortum1
1DepartmentofBioengineering,RiceUniversity
2DepartmentofMolecularandCellularOncology,TheUniveristyofTexasM.D.AndersonCancerCenter
Correspondenceto:[email protected]
URL:http://www.jove.com/details.php?id=2306
DOI:10.3791/2306
Keywords:Bioengineering,Issue47,Opticalimaging,intravitalmicroscopy,invivomicroscopy,endoscopicmicroscopy,fiberbundle,
DatePublished:11/1/2011
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,
andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Citation:Pierce, M.,Yu, D.,Richards-Kortum, R. High-resolutionFiber-opticMicroendoscopyforinsituCellularImaging.J.Vis.Exp.(47),e2306,
DOI:10.3791/2306(2011).
Abstract
Manybiologicalandclinicalstudiesrequirethelongitudinalstudyandanalysisofmorphologyandfunctionwithcellularlevelresolution.
Traditionally,multipleexperimentsareruninparallel,withindividualsamplesremovedfromthestudyatsequentialtimepointsforevaluationby
lightmicroscopy.Severalintravitaltechniqueshavebeendeveloped,withconfocal,multiphoton,andsecondharmonicmicroscopyall
demonstratingtheirabilitytobeusedforimaginginsitu1.Withthesesystems,however,therequiredinfrastructureiscomplexandexpensive,
involvingscanninglasersystemsandcomplexlightsources.Herewepresentaprotocolforthedesignandassemblyofahigh-resolution
microendoscopewhichcanbebuiltinadayusingoff-the-shelfcomponentsforunderUS$5,000.Theplatformoffersflexibilityintermsofimage
resolution,field-of-view,andoperatingwavelength,andwedescribehowtheseparameterscanbeeasilymodifiedtomeetthespecificneedsof
theenduser.
Weandothershaveexploredtheuseofthehigh-resolutionmicroendoscope(HRME)ininvitrocellculture2-5,inexcised6andlivinganimal
tissues2,5,andinhumantissuesinvivo2,7.Usershavereportedtheuseofseveraldifferentfluorescentcontrastagents,includingproflavine2-4,
benzoporphyrin-derivativemonoacidringA(BPD-MA)5,andfluoroscein6,7,allofwhichhavereceivedfull,orinvestigationalapprovalfromthe
FDAforuseinhumansubjects.High-resolutionmicroendoscopy,intheformdescribedhere,mayappealtoawiderangeofresearchersworking
inthebasicandclinicalsciences.Thetechniqueoffersaneffectiveandeconomicalapproachwhichcomplementstraditionalbenchtop
microscopy,byenablingtheusertoperformhigh-resolution,longitudinalimaginginsitu.
VideoLink
Thevideocomponentofthisarticlecanbefoundathttp://www.jove.com/details.php?id=2306
Protocol
1. MicroendoscopeAssembly
Thehigh-resolutionmicroendoscopedescribedhere(figure1a)shouldbeconsideredasabaseconfigurationwithseveralvariationspossiblein
assemblyandapplication.Wedescribeindetailhereanembodimentoftheplatformwhichisdesignedtobeusedwithproflavineasafluorescent
contrastagent.Proflavineisabrightnuclearstainwithpeakabsorptionandemissionwavelengthsof445nmand515nmrespectively.Theuseof
othercontrastagentswillrequiretheusertoselectexcitation,emission,anddichroicfiltersappropriately.Severalelementsofthehigh-resolution
microendoscopearegenericandmaybeobtainedfrommultiplevendors.Forexample,optomechanicalpositioningcomponentsareavailable
fromThorlabs,Newport,Linosamongothers.CompactCCDcamerasareavailablefromcompaniesincludingPointGreyResearch,Prosilica,
andRetiga;camerasensitivityshouldbechosenwithconsiderationofthebrightnessofthefluorophoretobeused,aswellasthedesiredframe
rate.High-powerlightemittingdiodes(LEDs)maybeobtainedfromLuxeon,Cree,andNichiaamongothers.Fiber-opticbundlesareavailable
fromSumitomo,Fujikura,andSchott.Inselectingcomponentsforaspecificapplication,theusershouldconsidertheinherentrelationships
involvedinfluorescencemicroscopybetweenfluorophoreconcentration,photobleaching,illuminationintensity,camerasensitivity,gain,and
exposuretime.
1. Connectthe6"cagerodstothe1.5"cagerods,toformapairof7.5"longrods.Screwtheserodsintoonefaceofthefoldmirrorunit.Screw
the0.5"rodsintotheoppositeface.Slidethecagecubeontothecagerods,viathetwolowerthroughholes.Screwthe2"rodsintotheside
faceofthecagecube(figure1b).
2. AttachthecameratoacageplatebyusingaC-mounttoSM1adapter.Securethecageplateonthe0.5"cagerods,flushwiththefaceofthe
foldmirrorunit.
3. Insertthe"tube"lensintoa3"longlenstubeandsecurethelenswitharetainingring.Thefocallengthofthelensshouldbeselectedsuch
thatthecoresofthefiber-opticbundlearesampledbyatleasttwopixelswhenprojectedontothecamera.Droptheemissionfilterintothe
tubeontopofthefirstretainingring,andaddanotherringtosecurethefilterinplace.Observethefilterorientationconventionindicatedby
thefiltermanufacturer.ScrewthelenstubeintothesideofthecagecubenearesttotheCCDcamera.Notethatinfigure1c,thislensand
filterareshownwithoutthe3"lenstubeforclarity.
Copyright©2011 CreativeCommonsAttributionLicense January2011| 47 |e2306|Page1of4
JournalofVisualizedExperiments www.jove.com
4. Connectthecameratoacomputerandviewtheimageonscreen.Directthecageassemblyatadistantobjectandslidethecagecubealong
therailsuntilanimageappearsinfocus.Lockdownthecagecubeatthislocation.(Thisisasimplemethodofensuringthatthetubelenswill
formafocusedimagewhencombinedwiththeinfinity-correctedobjectivelens).
5. Insertthedichroicmirrorintotheholderandplaceat45°inthecagecube.
6. Screwtheobjectivelens,viaaRMStoSM1threadadapterandanadjustablelenstube,intothefaceofthecagecubeoppositethetubelens
holder.Az-translatormaybeusedinplaceoftheadjustablelenstubeforeasierfocusing.ScrewanSMAconnectorintoacageplateand
mountthisontherods,approximatelyattheworkingdistanceoftheobjectivelens.
7. MountanLEDonacageplateandslideontotheendofthe2"rods.Addalenstoa0.5"lenstubesuchthatwhenthistubeisscrewedinto
thesideofthecagecube,itwillformanimageoftheLEDthatfillsthebackapertureoftheobjectivelens.This(Kohlerillumination)
configurationwillensurethattheproximalfaceofthefiber-opticbundleisuniformlyilluminated.Addtheexcitationfiltertothe0.5"lenstube
andsecureinplacewitharetainingring(figure1c).
8. AttachaSMAconnectortoafiber-opticbundle.ScrewtheSMAconnectorizedbundleintotheSMAreceptaclemountedonthecagerods.
Directthedistalendofthebundletowardsabroadbandlightsource(fluorescentlightingwillsuffice)andobservetheimageofthebundle
proximalfaceontheCCDcamera.Adjustthepositionoftheobjectivelensbyscrewinginoroutofthecagecubeuntilthefiberbundleimage
appearsinfocus.Theindividualcoresshouldbeclearlyvisible(Figure2).
2. GRIN LensAssembly
Thespatialresolutionofthemicroendoscopecanbeincreasedbyattachingamicro-lensorlensassemblytothedistaltipofthefiberbundle.
Theseopticsareconfiguredsuchthatinsteadofplacingthebundletipdirectlyontothetissue,thetipisimagedontothetissuesurfacewith
demagnification,therebyincreasingthespatialsamplingfrequencyimposedbythelight-guidingcoresofthefiberbundle.Thedegreeof
demagnificationcorrespondstotheincreaseinspatialresolution,andatthesametime,toaproportionatedecreaseinfield-of-view.Gradient
index(GRIN)lenscomponentsarecompatiblewithfiber-opticsandareavailablefromGrinTech,NSG,Schott,amongothers,andcanbedirectly
bondedtothedistaltipofafiberbundle.
1. SelectaGRINlenswiththedesiredmagnificationandworkingdistanceforyourapplication.Ensurethatthediameterofthelensexceeds
thatofthefiberbundleyouplantoworkwith.WerecommendthatthefiberbundleandGRINlensbemountedonseparate3-axismanual
positioningstagesunderalowpowermicroscopeorstereoscopeforaccuratealignmentpriortobonding.
2. Placeadropofopticaladhesive(eg.NorlandUVcuringadhesive)oneitherthelensorbundleface.Bringthetwocomponentsintocontact
byusingthemanualpositioners.ExposetheinterfacetoUVlightforthedoserecommendedbythemanufacturer.
3. ToprotecttheGRINlensandthebondedinterface,ashortlengthofaluminumcapillarytubing(SmallPartsInc.)canbeusedtoenclosethe
joint.Slidethetubingoverthejointandsecureinplacewithepoxy.Heatshrinktubingcanbeusedtofinishtheassembly.
3. Microendoscope Imaging
1. Applythecontrastagenttothecellsortissuetobeimaged.Withproflavine(0.01%w/vinPBS),invitroimagingofcellsinculturecanbe
performedbybriefincubation(<1minute)andthoroughrinsing.Imagingofexvivotissuespecimensorinvivotissuesispossiblefollowing
topicalapplicationofthedye.Uptakeofproflavineundertheseconditionshasbeenfoundtobenear-instantaneous,withimagingpossible
withinafewsecondsandlastingforseveralminutes.
2. Placethefiberopticbundleinlightcontactwiththesampletobeimaged.Whenimagingcellsincultureorexvivotissuespecimens,we
recommendmountingthedistalendofthefiberbundleinasecurefixturewithmanualpositioningstagesonXYZaxesforstabilityduring
imaging.
4. Representative Results:
Whenassembledcorrectly,themicroendoscopewilloperateasanepi-fluorescencemicroscope,relayedthroughacoherentfiber-opticbundle.
Foroptimalimagingresults,attentionshouldbepaidtoensuringthatthreekeyconditionsaremet:
1. TheproximalfaceofthefiberbundleshouldbeimagedontotheCCDcamerawithoutdefocus,inordertoachievethefullresolutionofthe
system.Figure2a,b,cdemonstrateaportionofafiberbundleimagedwithpoorfocus,slightdefocus,andgoodfocus,respectively.The
optimumfocusisfoundbyadjustingtheaxialpositionoftheobjectivelensrelativetothebundleface.
2. Theproximalfaceofthefiberbundleshouldbeuniformlyilluminatedoveritsfulldiameter(field-of-view).AsdescribedintheProtocolText
(1.7),thisisachievedbyconfiguringtheilluminationopticsforKohlerillumination.Figure2dshowsanimageacquiredwhenauniform
fluorescentsampleisilluminatedinthispreferredconfiguration,withFigure2edemonstratingthecorrespondingresultundercritical
illumination.Inthelattercase,thestructureoftheLEDisimagedontothefiberbundleface,resultingintheappearanceofthisunwanted
patternsuperimposeduponthetruesamplestructure.
3. Boththeproximalanddistalfacesofthefiberbundleshouldbecleanandfreeofscratchesandchips.Figure2fdemonstratesthepresence
ofdebrisattachedtothebundleface,withdamageintheformofasmallchipat5o'clockonthefiberperimeter.Debriscanberemovedfrom
thebundlefacesbycleaninginthesamefashionasconventionalfiber-opticconnectors,withlenspaperandisopropanol,orstandard
fiber-opticcleaningtools.Ifeitherendofthefiberbundleisscratchedorchipped,orifthebundlebreaksalongitslength,thefacemaybe
polishedflatbystandardmanual,ormechanicalpolishingtechniques.Werecommend12-15μmlappingpaperforaninitialcoarsepolish,
withafinalpolishon0.5-1.0μmpaper.
Figure3ademonstratesimagingof1483cellsinvitro,followinglabelingwithproflavineandlightplacementofthebarefiberbundleonthe
sample.Figure3bdemonstratestheimprovementinspatialresolutionandreductioninfield-of-viewprovidedbya2.5xGRINlensbondedtothe
bundletip.Movie1demonstratesinvivoimagingofthemammaryfatpadinamousemodel.Here,afiberbundlewith0.5mmouterdiameter
(330μmfield-of-view)waspassedthrougha21-gaugeneedleandadvancedintothetissue.Fatcellsareclearlyvisible,withmotionduetothe
cardiaccycleapparentinthisacquisitionat15framespersecond.Figure3cdemonstratesimagingoftheoralmucosainahealthyhuman
volunteer,thistimeusingalargerfiberbundlewith1.5mmouterdiameter(1.4mmfield-of-view).Inallexamplesshown,proflavinewasusedas
anuclearlabelingfluorescentcontrastagent.
Copyright©2011 CreativeCommonsAttributionLicense January2011| 47 |e2306|Page2of4
JournalofVisualizedExperiments www.jove.com
Figure1.Assemblingthehigh-resolutionmicroendoscope(HRME).(a)SchematicdiagramoftheHRMEsystem.(b)Assemblyofthemain
optomechanicalsupportstructure.(c)Additionofopticalelements,illuminationLED,andCCDcamera.(d)PhotographoftheHRMEsystem,
packagedina10"x8"x2.5"enclosure.
Figure2.SettinguptheHRME.Examplesofimagingwiththefiber-opticbundlein(a)poorfocus,(b)closetogoodfocus,(c)idealfocus.In(d),a
uniformfluorescenttargetatthebundle'sdistaltipisimagedunderKohler(uniform)illumination.(e)Auniformfluorescenttargetimagedunder
criticalillumination,withthesourcestructureapparentontheobject.(f)Loosetissueandcellscansticktothefiberbundleface,whichisalso
pronetominordamageatitsperiphery.
Figure3.ImagingwiththeHRME.(a)1483cellsinvitro,imagedwithabarefiberbundle(IGN-08/30)followinglabelingwithproflavine0.01%
(w/v).(b)Thesame1483cellcultureasshownin(a),imagedwithafiberbundlewith2.5xGRINlensattached.(c)Imageofnormalhumanoral
mucosainvivo,followingtopicalapplicationofproflavine0.01%(w/v).
Movie1.Imagingthemammaryfatpadofamouseviainsertionofa450μmouterdiameterfiberbundlewithinthelumenofa21-gaugeneedle
passedintothetissue.Proflavine0.01%(w/v)wasdeliveredtotheimagingsitethroughthesameneedlepriortoinsertionoftheimagingfiber.
Clickheretowatchvideo
Copyright©2011 CreativeCommonsAttributionLicense January2011| 47 |e2306|Page3of4
JournalofVisualizedExperiments www.jove.com
Discussion
Thehigh-resolutionmicroendoscopytechniquedescribedhereprovidesresearchersinthebasicbiomedicalandclinicalresearchareaswitha
flexible,robust,andcost-effectivemethodforvisualizingcellulardetailinsitu.Wehavedescribedaprotocolforassemblingtheimagingsystem
anddemonstrateditsuseincellcultureinvitro,andinanimal,andhumantissuesinvivo.Whiletheimagingresultspresentedhereused
proflavineasafluorescentcontrastagent,othergroupshavedemonstratedversionsofthesystemwithLEDilluminationwavelengthsandfilters
chosentomatchexcitation/emissionspectraofotherdyes5-7.
Resolutionandfield-of-viewareinitiallydeterminedbythecore-to-corespacingandimagingdiameterofthefiber-opticbundle.Wehaveused
bundleswithapproximately4μmcore-corespacing,andimagingdiametersof330μm(movie1),720μm(Figure2,Figure3a,b),and1400μm
(Figure3c).Thesmallerbundlescanbepassedthroughnarrowergaugeneedlesandaresignificantlymoreflexiblethanthelargerfibers.Weand
others8have,insomecases,notedtheappearanceofautofluorescenceemissionsfromthefiberbundleitself.Whenattemptingtoexcite
fluorophoresatUVwavelengths,orcollectemissionintheredspectralrange,attentionshouldbepaidtotheleveloffiberbundle
autofluorescencecontributingtotheoverallmeasuredsignal.
Whilemostofthehigh-resolutionmicroendoscopyworkreportedto-datehasusedabarefiberbundle,additionalmagnificationcanbeprovided
byuseofGRINlensesbondedtothedistaltip.GRINlensesofferastraightforwardandeconomicalwaytoincreasespatialresolution,though
theirsusceptibilitytoopticalaberrationsandlimitedNAiswellrecognized.IfGRINlensperformanceisinadequateforaparticularapplication,
hybridGRIN/sphericallensobjectives9orminiatureobjectivelensassemblies10-11canbeemployed.
Thehigh-resolutionmicroendoscopedescribedhereessentiallyoperatesasawide-fieldepi-fluorescencemicroscope;thereforenooptical
sectioning(asinconfocalornonlinearmicroscopy)istobeexpected.Inourexperience,using455nmexcitationlightandtopicalproflavineasa
contrastagent,lightisprimarilycollectedfromadepthcorrespondingtoafewcelllayers.
Thisprotocoloughttoenablethereadertoassemblethehigh-resolutionmicroendoscopeonthebenchtop,withacompactfootprintof10"x8".If
desired,thesystemmaybeenclosedinaboxandtheelectricalcomponents(LEDandcamera)poweredbyabatterypack(Figure1d).Many
compactcamerascanbepoweredbytheIEEE-1394(Firewire)andUSBportsofthehostcomputer.
Disclosures
MPandDYhavenothingtodisclose.RRKholdspatentsrelatingtomicroendoscopicimagingplatforms.
Acknowledgements
ThisresearchwaspartlyfundedbytheNationalInstitutesofHealth,grantR01EB007594,theDepartmentofDefenseBreastCancerResearch
Program,proposalBCO74699P7,andtheSusanG.KomenFoundationgrant26152/98188972.
References
1. Pierce,M.C.,Javier,D.J.,Richards-Kortum,R.Opticalcontrastagentsandimagingsystemsfordetectionanddiagnosisofcancer,Int.J.
Cancer123,1979-1990(2008).
2. Muldoon,T.J.,Pierce,M.C.,Nida,D.L.,Williams,M.D.,Gillenwater,A.,Richards-Kortum,R.Subcellular-resolutionmolecularimaging
withinlivingtissuebyfibermicroendoscopy.Opt.Express15,16413-16423(2007).
3. Muldoon,T.J.,Anandasabapathy,S.,Maru,D.,Richards-Kortum,R.High-resolutionimaginginBarrett'sesophagus:anovel,low-cost
endoscopicmicroscope.Gastrointest.Endosc.68,737-744(2008).
4. Muldoon,T.J.,Thekkek,N.,Roblyer,D.,Maru,D.,Harpaz,N.,Potack,J.,Anandasabapathy,S.,Richards-Kortum,R.Evaluationof
quantitativeimageanalysiscriteriaforthehigh-resolutionmicroendoscopicdetectionofneoplasiainBarrett'sesophagus.J.Biomed.Opt.15;
026027(2010).
5. Zhong,W.,Celli,J.P.,Rizvi,I.,Mai,Z.,Spring,B.Q.,Yun,S.H.,Hasan,T.Invivohigh-resolutionfluorescencemicroendoscopyforovarian
cancerdetectionandtreatmentmonitoring.Br.J.Cancer101,2015-2022(2009).
6. Dubaj,V.,Mazzolini,A.,Wood,A.,andHarris,M.Opticfibrebundlecontactimagingprobeemployingalaserscanningconfocalmicroscope.
J.Microsc.207,108-117(2002).
7. Dromard,T.,Ravaine,V.,Ravaine,S.,Lévêque,J-L.,Sojic,N.Remoteinvivoimagingofhumanskincorneocytesbymeansofanoptical
fiberbundle.Rev.Sci.Inst.78,053709(2007).
8. Udovich,J.A.,Kirkpatrick,N.D.,Kano,A.,Tanbakuchi,A.,Utzinger,U.,Gmitro,A.F.Spectralbackgroundandtransmissioncharacteristicsof
fiberopticimagingbundles,Appl.Opt.47,4560-4568(2008).
9. Barretto,R.P.J.,Messerschmidt,B.,Schnitzer,M.J.Invivofluorescenceimagingwithhigh-resolutionmicrolenses,Nat.Methods6,511-512
(2009).
10. Rouse,A.R.,Kano,A.,Udovich,J.A.,Kroto,S.M.,Gmitro,A.F.Designanddemonstrationofaminiaturecatheterforaconfocal
microendoscope,Appl.Opt.43,5763-5771(2004).
11. Kester,R.T.,Christenson,T.,Richards-Kortum,R.,Tkaczyk,T.S.Lowcost,highperformance,self-aligningminiatureopticalsystems,Appl.
Opt.48,3375-3384(2009).
Copyright©2011 CreativeCommonsAttributionLicense January2011| 47 |e2306|Page4of4