Photoacoustic imaging of human coronary atherosclerosis in two spectral bands

(1)

Krista

Jansen

a,b,1

,

Min

Wu

a

,

Antonius

F.W.

van

der

Steen

a,b,c

,

Gijs

van

Soest

a,

*

a

DepartmentofBiomedicalEngineering,ErasmusMC,P.O.Box2040,3000CARotterdam,TheNetherlands

b_{Interuniversity}_Cardiology_Institute_of_The_Netherlands_–_Netherlands_Heart_Institute,_P.O._Box_19258,₃₅₀₁_DG_Utrecht,_The_Netherlands c

DepartmentofImagingScienceandTechnology,DelftUniversityofTechnology,Lorentzweg1,2628CJDelft,TheNetherlands

1. Introduction

Myocardialinfarctionisaleadingcauseofdeathworldwide[1].

In themajority of cases,they arecaused bythe ruptureof an

atheroscleroticplaqueandthesubsequentreleaseofits

thrombo-geniccontentintothebloodstream[2].Thepresenceofalipidrich

necroticcoreisoneofthedeterminantsofthesusceptibilityofa

plaque to rupture [3,4]. For that reason, the identiﬁcation of

necrotic core is a highly coveted imaging target. Intravascular

ultrasound (IVUS) radiofrequency data analysis techniques for

tissuecharacterization(VH-IVUS,iMap)havebeendeveloped,but

theiraccuracyandmutualconsistencyarestillunderinvestigation

[5–7].Nearinfraredspectroscopy(NIRS)incombinationwithIVUS,

canidentifythepresencebutnottheamountorlocation,relative

tothelumen,ofthelipidcore[8–10].

Intravascularphotoacoustic(IVPA)imaginghasdemonstrated

theabilitytodirectlyimagetissuecomponentsinthevesselwall,

withhighchemicalspeciﬁcityforlipidtype.Itutilizesdifferences

intheabsorptionspectraofthevesselwallconstituentstoidentify

tissuetypes.Effortshaveprimarilyconcentratedonlipiddetection,

andstartedinthevisiblewavelengthrange.Withtheintroductionof

suitablelightsources,focusshiftedtothenear-infraredwavelength

range,wherehemoglobinabsorptionismuchlower,allowingfor

betterlightpenetration.Inthiswavelengthrange,theabsorption

spectraoflipidsarecharacterizedbytwoprominentfeaturesaround

1.2and1.7

mm.

Theseabsorptionbandsaretheresultofthesecond

and ﬁrstovertones ofthe C–H bondvibrations within the lipid

molecules, respectively. The 1.2

mm

absorption band has been

exploitedextensivelytodistinguishlipidsfromhealthyvesselwall,

inrabbit[11,12]aswellashuman[13–15]atheroscleroticarteries.

Inrecentyears,lipiddetectionusingexcitationwavelengthsaround

1.7

mm

hasseenincreasedinterest[13,16–18].Inthiswavelength

range, the higher lipid absorption possibly leads to increased

sensitivityusinglowerlightintensity.However,waterabsorptionis

highertoo,whichcouldpotentiallyoffsettheincreasedsensitivity

forlipidsbylimitingthepenetrationdepth(Fig.1).

Both absorption bands each consist of several overlapping

peaks as a result of C–H bond vibrations within the different

structuralgroups(–CH3,>CH2,BBCHand>CH(aromatic))ofthe

lipidmolecules[19–21].The positionand relativeheightofthe

peaks vary with the number and location of these different

structural groups within the molecules and therefore provide

chemical speciﬁcity. The possibility for differentiating between

plaque lipids and peri-adventitial lipids, based on the speciﬁc

ARTICLE INFO

Articlehistory:

Received18September2013

Receivedinrevisedform29October2013 Accepted16November2013 Keywords: Intravascularimaging Atherosclerosis Vulnerableplaque Tissuecharacterization Spectroscopy Lipids ABSTRACT

Spectroscopicintravascularphotoacousticimaging(sIVPA)hasshownpromisetodetectanddistinguish lipidsinatheroscleroticplaques.sIVPAgenerallyutilizesoneofthetwohighabsorptionbandsinthe lipid absorption spectrum at 1.2mm and 1.7mm. Speciﬁc absorption signatures of various lipid compounds withinthe bandsin eitherwavelength range can potentially be usedto differentiate betweenplaquelipidsandperi-adventitiallipids.Withtheaimtoquantifyanydifferencesbetweenthe twobands,weperformedcombinedsIVPAimaginginbothabsorptionbandsonavesselphantomandan atherosclerotichumancoronaryarteryexvivo.Lipiddetectioninahumanatheroscleroticlesionwith sIVPArequiredlowerpulseenergyat1.7mmthanat1.2mm(0.4mJversus1.2mJ).Theimagingdepth wastwiceaslargeat1.2mmcomparedto1.7mm.Adequatedifferentiationbetweenplaqueand peri-adventitiallipidswasachievedat1.2mmonly.

ß2013TheAuthors.PublishedbyElsevierGmbH.Allrightsreserved.

§

This isanopen-access articledistributedunderthetermsoftheCreative CommonsAttributionLicense,whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

* Correspondingauthor.Tel.:+31107044638;fax:+31107044720. E-mailaddress:g.vansoest@erasmusmc.nl(G.vanSoest).

1

Presentaddress:DepartmentofENT/Audiology,VUMedischCentrum,P.O.Box 7057,1007MBAmsterdam,TheNetherlands.

2213-5979/$–seefrontmatterß2013TheAuthors.PublishedbyElsevierGmbH.Allrightsreserved.

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absorptionsignatureofthevariouslipidcompounds,remainstobe explored.

Theseconsiderationsoutlinetradeoffsintermsofsensitivity,

imagingdepth,andpossiblychemicalspeciﬁcityconnectedtothe

choiceofIVPAwavelength.Inthispaper,wepresentspectroscopic

IVPA(sIVPA)imagingofalipidcontainingvesselphantomandan

atherosclerotic human coronary artery ex vivo at 1.2

mm

and

1.7

mm,

providingadirectcomparison betweenthewavelength

ranges.In thephantom,weacquiredhigh-resolutionspectraof

cholesterol,cholesterololeateandcholesterollinoleate,

represen-tativeofplaquelipids,and peri-adventitialtissue.Theresulting

spectrawereusedtodeterminealimitednumberofwavelengths

thatmaximizethedifferencebetweenplaqueandperi-adventitial

lipids.At these wavelengths, we obtained co-registered sIVPA/

IVUSimages ofthevesselphantomthat weused todetectthe

plaqueand peri-adventitiallipidsalternatively.Withtwo

wave-lengthsperspectralrangeonly,thelipiddetectioncapabilityin

eachrangewasexaminedusingboththephantomdataandtheex

vivodataofadiseasedhumancoronaryarteryspecimen.

2. Methods

2.1. Phantomdesign

Todeterminethecapacityforlipiddetectionanddifferentiation

at1.2and1.7

mm,

wemadeacylindricalvesselmimickingphantom

(Fig.1a). Thephantomconsisted of10% (byweight)

poly-vinyl-alcohol (PVA) crystals in demineralized water that formed an

acousticallytransparent gel after2 freeze/thaw cycles.It had a

central lumen with a diameter of 3mm and four 5mm deep

cylindricalcavitieswithadiameterof1.5mm,locatedat500

mm

fromthelumen.Weﬁlledthreecavitieswithcholesterol,cholesterol

oleateandcholesterollinoleate(SigmaAldrichCo.,C8667,C9253

andC0289,resp.). Thesearethe threemost abundantlypresent

lipids in atherosclerotic lesions [23,24], and are assumed to be

representativeofplaquelipids.Thefourthcavitywasﬁlledwith

peri-adventitialtissuethatwasobtainedfromahumancoronary

arteryspecimen,seedescriptionbelow.Inperi-adventitialtissue,

lipidsaredepositedasamixtureoffattyacids[25].

2.2. Humanarteryacquisitionandhandling

Ahumancoronaryarterywascollected atautopsy fromthe

Department of Pathologyof the ErasmusMedical Center(MC),

after obtainingconsent from therelatives and approval of the

researchprotocolbytheMedicalEthicsCommitteeoftheErasmus

MC(MEC-2007-081).Thecoronaryarterywasfrozenwithin4hat

808Cand stored. Itwasthawed and measured three months

later.

2.3. Combinedintravascularphotoacousticandultrasoundimaging

system

All co-registered sIVPA/IVUS images were acquired using a

combinedIVPA/IVUS imaging systemdescribedpreviously[14].

Theexcitationlightforphotoacousticimagingwassuppliedbya

tunablelaser(OPOTEKVibrantB/355-II)withapulsedurationof

5nsandarepetitionrateof10Hz.Thelaserwascoupledtothe

custom-built catheter by a tapered multimode ﬁber (Oxford

Electronics, Four Marks, UK; input diameter 1mm; output

diameter360

mm).

ThehybridIVPA/IVUScatheterprototypeweusedissimilarto

those used earlier [14], but with a different transducer. It

compriseda400

mm

diametercoreopticalﬁber(PioneerOptics,

Bloomﬁeld,CT)todeliverthelightpulsestothevesselwall.The

ﬁbertipwaspolishedundera348anglecoveredbyaquartzcapto

maintain an air–glass interface deﬂecting the beam by total

reﬂection. Anultrasoundtransducerwasplaced distalfromthe

ﬁber tipto transmitand receiveultrasound waves. The 0.4 by

0.4mm lead magnesium niobate-lead titanate(PMN-PT) single

crystalultrasoundtransducerwasdesignedandcustombuiltby

the Departmentof BiomedicalEngineering ofthe University of

SouthernCalifornia[26]andhadacenterfrequencyof44.5MHz

anda 6dBfractionalbandwidthof45%.Theseparationbetween

ﬁber tip and transducer center was approximately 1mm; the

opticalandacousticalbeamoverlappedbetween0.5and4.5mm

from the transducer, with an angle of 228. The catheter tip

assemblyhadanouterdiameterof1mm.

The catheter was rotated using a motorized rotary stage

(SteinmeyerGmbH&Co.KG).Forpulseechoimaging,anarbitrary

waveformgenerator(TaborElectronicsWW2571A)transmitteda

Gaussian-modulatedcosinewave whichwastransmittedtothe

probethroughacustom-builtexpanderandlimiter.ReceivedUS

and PA signals wereband pass ﬁltered (13–60MHz 5th order

Butterworth,custombuilt),ampliﬁedbya43dBampliﬁer(Miteq

AU1263)anddigitizedatasamplefrequencyof350MSs 1_by_a

12-bitdataacquisitioncard(AcqirisDP310).

2.4. Phantommeasurements

Usingthedualmodalityimagingsystemdescribedabove,we

imagedthelipidcontainingvesselphantominawaterbath,with

thecombinedIVPA/IVUScatheterpositionedinthelumen.Weﬁrst

acquiredacrosssectionalIVUSimagetolocatethelipidinclusions.

Fig.1.(a)Lipidandwaterabsorptioninthenear-infraredwavelengthregion,showingthetwohighpeaksinthelipidabsorptionspectrumaround1210and1720nm.Inthese twoopticalwindows,lipidabsorptionexceedswaterabsorption.Lipidabsorptionat1720nmis5.5timeshigherthanat1210nm;waterabsorptionis5timeshigher. Adaptedfrom[22].(b)Transmissionoflightthroughwater;computedbasedondatafromhttp://omlc.ogi.edu/spectra/water/abs/index.html.

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afterwhich2wavelengthswereaddedtocreateafairlydistributed spacing.

Next, we obtained two-dimensional spatially co-registered

spectroscopicIVPAandIVUSimagesofthephantomatthese12

wavelengths(6perwavelengthrange)byrotatingthecatheterin

18stepsandacquiringphotoacousticandultrasoundimagelinesat

everystep.Ateveryangle,thelaserwastunedthroughthespectral

rangeofinterest(1.2and1.7

mm)

toensureco-registrationofthe

IVPAdataatallwavelengths.Forultrasoundpulseechoimaging,

wetransmitteda10VpeaktopeakGaussian-modulatedcosine

wave with a center frequency of 44.5MHz and a 50% 6dB

bandwidthrelative tothe peak.IVUSimages wereobtained by

averagingtheechoesfrom8transmissionsperline;IVPAwasnot

averaged(onelaserpulseperwavelengthperimageline).

2.5. Arterymeasurements

The human coronary artery was placed in a TPX (TPX1

Polymethylpentene)holderwith200

mm

thickmetalwiresglued

atevery1.5mmperpendiculartothelongitudinalaxistoprovide

imageregistration.Theholder wasthen placedin awater tank

containingasalinesolutionatroomtemperature.Thearterywas

tiedonacannulathroughwhichthecatheterwasintroduced.To

ﬁnd sites of interest, we performed an IVUS pullback using a

commercialIVUSsystem(BostonScientiﬁc iLab,AtlantisSRPro

catheters),usingthemetalwiresasreferencepoints.Theselected

wireswerethenfoundusingourcombinedIVPA/IVUScatheter.

Spatiallyco-registeredsIVPA/IVUScrosssectionalimageswere

obtained by rotation of thecatheter in 18 steps and acquiring

photoacoustic and ultrasound image lines at every step. Two

rotationswereperformed toobtaintwoco-registeredimagesat

1205and1235nm,andat1680and1710nm,separately.Atevery

angle, thelaser was tuned from 1205 to 1235nm in the ﬁrst

measurement, and from 1680 to 1710nm in the second

measurement, to ensure co-registration of the IVPA data per

wavelength range. The ultrasound signal transmitted for pulse

echoimaging,aswellasthenumberofaveragingandtheaverage

pulseenergy,werethesameasinthephantommeasurement.

2.6. IVPAspectraldataprocessing

ThedigitizedspectroscopicIVPAdataofthefourlipidinclusions

inthephantom,werebandpassﬁlteredbetween10and70MHz

usinga100thorderzero-phaseforwardandreverseﬁniteimpulse

response(FIR)ﬁlter,andsubsequentlyupsampled,correctedfor

jitteranddownsampledtotheoriginalsamplingfrequency.Next,a

Tukeywindowandenvelopeﬁlterwereapplied.Acorrectionfor

variationsinthelightenergywasemployed,usingtheamplitude

of the signal close to the transducer, which is caused by the

absorption of laser pulses in the ultrasound transducer and

cathetertip.Depthlocationsofhighsignalintensitywerechosen

byselectionofallpeaksaboveacertainthresholdinthe1205and

1710nmenvelopedsignaltraces,respectively.Spectraatselected

oflaser pulsesin theultrasoundtransducerand cathetertip.It

presents in the IVPA image as bright rings, concealing the

photoacoustic signals produced by the arterial tissue close to

thecatheter.AsimilarcircularartifactintheIVUSdata,causedby

the‘ringing’ofthetransducerasaresultofthetransmissionof

ultrasoundpulses,wasremovedbysubtractingthemeaninthe

angulardirectionoftheaffectedpartofthedata.BoththeIVPAand

IVUSdatawerethen Tukeywindowedand envelopeﬁltered.A

correction forvariationsin thelight energybetween individual

pulsesandbetweenthedifferentwavelengths,usingthe

ampli-tudeintheringartifactmentionedabove,wasappliedtotheIVPA

data.Wesubsequentlyscan-convertedtheIVPAandIVUSdatato

Cartesiancoordinatesandlogcompressedthemfordisplay.The

‘hot’and‘gray’colormapsinMatlab(R2007b)wereusedforthe

IVPAandIVUSimages,respectively.TocreatecombinedIVPA/IVUS

images, weoverlaidtheIVPA data on theIVUSimages usinga

nonlinearred-yellow-whitecolorscaleandalineartransparency

scale.AlldataprocessingwasdoneusingMatlab(R2007b).

2.8. sIVPAdataanalysisforlipiddifferentiationanddetection

Toinvestigate thecapability ofsIVPAin thetwo absorption

bandstodistinguishplaquefromperi-adventitiallipids,thetwo

6-wavelengthsIVPAdatasetsofthelipidcontainingvesselphantom

wereprocessed as described above,up to scan-conversion.For

eachpixelintheresultingdatasets,thecorrelationcoefﬁcientRof

thePAspectrumwithtworeferencespectrawascomputed.We

used thePA spectra ofcholesterol and peri-adventialtissue as

referencespectrafor plaque and peri-adventitial lipids,

respec-tively.Oftheplaquelipidspectra(Fig.2candd),ineitherspectral

range,thecholesterolspectrumhasthelowestcorrelationwiththe

peri-adventitial spectrum (Fig. 2b) and therefore is the most

suitabletodistinguishplaquefromotherlipids.

The6-wavelength correlationcoefﬁcientsRx(xis lipidtype;

‘‘chol’’ for cholesterol or ‘‘PL’’ for peri-adventitial lipids) were

medianﬁlteredover48intheangulardirectionand8samplesin

theradialdirection.Thethresholdvalues Rc,xforthecorrelation

coefﬁcientswerechosenempirically:thelowestvaluesforwhich

plaquelipidscouldstillbeseparatedfromperi-adventitiallipids

wereselected.Tocreatealipidmap,thelipidmatchingregions–

those witha correlationcoefﬁcientequal toorhigher thanthe

thresholdvalueRc,x–weredisplayedinredandoverlaidonthe

correspondingIVUSimage.

WecomparedthepotentialofsIVPAat1.2

mm

andat1.7

mm

forlipididentiﬁcationusingtwowavelengths,

l

h,vand

l

l,v(hfor

highlipidabsorption;lforlowlipidabsorption)perwavelength

range

v.

Wecomputedlipidmapsofthephantomandarterycross

sectionusingthefollowingalgorithm:wedeterminedthenoise

levelat

l

h,vbysamplingthePAsignalinsidethelumen(identiﬁed

intheIVUSimage)andmaskedoutallPAsignalbelowthatlevel.

Wethencalculatedtherelativedifference

d

v=[I(lh,v) I(ll,v)]/

I(lhv),whereI(l)isthePAsignalamplitudeatwavelength

l.

d

was

(4)

Lipidswereidentiﬁedby

d

>

d

c,where

d

carethethresholdvalues

determinedfromtheanalysisoftheabsorptionspectraofpure

lipids,seeFigs.2canddand5.

2.9. Histologicalvalidation

Afterimaging,wecutthearteryatthetwowiresadjacenttothe

imaging planetoobtaina 3mm thickarterysegmentwiththe

imagedcross-sectioninthemiddle.Thesegmentwasembeddedin

‘‘optimal cutting temperature’’ (OCT) compound (Tissue-Tek1

,

Sakura Finetek Europe B.V.), frozen in liquid nitrogen cooled

isopentanevapor,andstoredat 808Cuntilserialsectioningfor

staining.WeperformedOilRedO(ORO)stainingtoidentifylipids

(stained red). A Hematoxylin–Eosin (H&E) stain was used to

provideanoverviewofthearterycrosssection;aResorcin–Fuchsin

(RF)stainwasusedtodemonstratethemorphologyandﬁbrous

structureofthevesselcross-sections.

3. Results

3.1. Lipiddifferentiationinphantom

WeperformedsIVPAmeasurementsinthedirectionsindicated

bythe whitedashedlines in thephotograph(topview) of the

phantominFig.2a.ThedatawereanalyzedasdescribedinSection

2.6 and the resulting averaged normalized PA spectra of

cholesterol, cholesterol oleate, cholesterol linoleate and

peri-adventitialtissue in the 1.2 and 1.7

mm

wavelength range are

showninFig.2candd,respectively.Whilethespectraat1.2

mm

exhibitmainlydifferencesinrelativepeakheight,thedominant

differences at 1.7

mm

are shifts in the locations of the peaks

betweenthespectraofplaqueandperi-adventitiallipids.

We obtained cross sectional sIVPA/IVUS data of the

lipid-containing vessel phantom at 6 wavelengths in both spectral

ranges. The wavelengths are given in Table 1. The lipid maps

resultingfromthecorrelationofthedatainthe1.2

mm

rangewith

thecholesterolandtheperi-adventitiallipidreferencespectrum

aredisplayedinFig.3aandb,respectively;Thecorrespondinglipid

mapsobtained inthe1.7

mm

rangeareshownin Fig.3cand d,

respectively. All lipid maps are overlaid on the associated,

co-registered IVUSimage. At1.2

mm,

thecholesterol and the two

cholesterolesters,representativeofplaquelipids,arealldetected

clearly, while the peri-adventitial tissue remained concealed

(Fig.3a).At1.7

mm,

however,itwasnotpossibletosimultaneously

detectthecholesterololeateandkeeptheperi-adventitialtissue

invisible.Ontheotherhand,moreofthecholesterolwasdetected

thanat1.2

mm.

Thecorrelationwiththeperi-adventitialreference

spectrumresultedindetectionoftheperi-adventitialtissueinthe

1.2

mm

wavelength range, while suppressing the other lipids

(Fig. 3b). In the 1.7

mm

wavelength range, less of the

peri-adventitialtissuewasdetected(Fig.3d).

Table 2 lists the threshold correlationvalues Rc,x for the

6-wavelengthsat1.2and1.7

mm

thatwereusedtocreatethelipid

mapsinFig.3a–d.Tobeabletodetectperi-adventitiallipidswhile

Fig.2.LipidcontainingvesselphantomandIVPAspectraoflipidinclusions.(a)Photographofthephantom(top-view),ﬁlledwithcholesterol(bottom),cholesterololeate (right),cholesterollinoleate(top)andperi-adventitialtissue(left).(b)6-Wavelengthcorrelationcoefﬁcientsbetweenthespectraofthelipidinclusions.(c)Average, normalizedPAspectraofthefourlipidinclusionsinthe1.2mmwavelengthrange,and(d)inthe1.7mmwavelengthrange.c,cholesterol;cl,cholesterollinoleate;co, cholesterololeate;pl,peri-adventitiallipids.

Table1

Wavelengthsusedfor6-wavelengthlipiddetection. Wavelengthrange(mm) Wavelengths(nm)

1.2 1185,1195,1205,1215,1225,1235 1.7 1680,1710,1718,1726,1734,1751

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suppressingplaquelipids, thecorrelationcoefﬁcientshad tobe

chosenmuchhigherforthe1.7thanforthe1.2

mm

wavelength

range.

3.2. Lipiddetectioninphantomandartery

WeinvestigatedtheabilityofsIVPAtodetectlipidsusingtwo

wavelengths at 1.2

mm

versus the 1.7

mm,

in both the lipid

containingvesselphantomandahumancoronaryarteryexvivo.

The phantom results are shown in Fig. 4. The co-registered

combinedIVPA/IVUSimagesat1205(highlipidabsorption)and

1235nm (low lipid absorption) and the resulting relative

difference lipid map, are displayed in Fig. 4a–c, respectively.

Fig.4d–fdepictsthecorrespondinghighandlowlipidabsorption

imagesinthe1.7

mm

wavelengthrange,at1710and1680nm,and

lipidmap,respectively.Inbothwavelengthranges,allfourlipid

containingcavitiesexhibitanincreasedPAsignalat

l

hcompared

tothePAsignalat

l

l.Inthe1.2

mm

rangeIVPAimages,however,a

highersignalcanbeobservedfromnon-lipidregions,comparedto

the1.7

mm

range IVPA images, due tothe overall higher light

ﬂuence.Bothlipid mapssucceedequally wellindisplaying the

peri-adventitiallipidregionbuttheplaquelipidsweredisplayed

moreclearlyinthe1.7

mm

wavelengthrange.

The false positives in the 1.7

mm

lipid map at 2 times the

distanceofthelipidsfromthecatheteraretheresultofincomplete

suppression of the pulse echo signal that is generated by

absorptionof thelight in thetransducer and cathetertip. This

ultrasound signal is also visible in the 1.2

mm

range but is

effectivelysuppressed,aswellasthehigherPAbackgroundsignal.

Thethresholdvalues

d

ctorealizethelipidmapsinFig.4candf,

were chosen using the spectral data of the separate lipid

compounds.The5thto95thpercentileoftherelativedifference

ofthePAsignalathighandlowlipidabsorptionwavelengthsofthe

individual (unaveraged) IVPA spectra measured in the lipid

containing phantom were calculated (Fig. 5). The relative

differenceoftheabsorptioncoefﬁcientofelastinandcollagenat

thesewavelengths,isshownaswell.Thresholdvalueswerechosen

asthemidpointbetweenthelowestvaluefoundforthelipidsand

thehighestvaluefoundforelastinandcollagen(reddottedline).

Theresultingthresholdvalues

d

cinbothwavelengtharelistedin

Table2.

Theresultsof theatherosclerotic humancoronaryspecimen

(leftanteriordescendingartery,maleaged65)measurementare

showninFig.6.Thetoprow,Fig.6a–c,displaystheresultsobtained

inthe1.2

mm

wavelengthrange;thebottomrow,Fig.6d-f,the

resultsobtained usingwavelengthsaround1.7

mm.

Fromleftto

right,theIVPAimages at

l

h,theIVPA imagesat

l

l,andthe

2-wavelengthrelativedifferencelipidmapsareshown.Allimages

areoverlaidonthecorrespondingIVUSimage.TheIVUSimages

showasmalllumenwithaneccentricplaqueatthebottomrightof

the vessel wall. A large calciﬁcation is present in the plaque,

Fig.3.Lipidtypinginalipid-containingvesselphantomusingsIVPAat1.2and1.7mm.(a)Lipidmapbasedon6-wavelengthcorrelationwiththecholesterol,and(b)withthe peri-adventitialreferencespectruminthe1.2mmwavelengthrange(1185,1195,1205,1215,1225and1235nm).(c)Lipidmapbasedonthe6-wavelengthcorrelationwith thecholesterol,and(d)withtheperi-adventitialreferencespectruminthe1.7mmwavelengthrange(1680,1710,1718,1726,1734and1751nm).Alllipidmapsareshown overlaidonthecorrespondingIVUSimage(dynamicrange65dB).Plaquelipids,representedbycholesterol(bottom),cholesterololeate(right)andcholesterollinoleate(top) aredistinguishedclearlyfromperi-adventitialtissue(left)at1.2mm,whilelipidtypingat1.7mmyieldedaninferiorresult.

Table2

ThresholdvaluesRc,xanddc.

Thresholdvariable Wavelengthrange(mm)

1.2 1.7

Rc,chol 0.87 0.88

Rc,PL 0.88 0.96

(6)

indicatedbytheshadowingintheplaquearea.TheOROlipidstain,

depicted in Fig. 6g, conﬁrms these ﬁndings (lipids in red;

calciﬁcation black). The 1205nm IVPA image shows a slightly

enhancedsignalfromthelipidareainthetoppartoftheplaque,as

wellasastronglyenhancedsignalintheperi-adventitialregion

aroundthevesselwall,comparedtothe1235nmIVPAimage.In

the1.7

mm

range,theenhancementofthesignalinthelipidrich

plaqueareaismorepronouncedthaninthe1.2

mm

range,while

thesignalenhancementintheperi-adventitialregionis

compara-ble.Additionally, thecalciﬁed regionproduces muchless signal

around1.7

mm,

albeitstillinthesameorderofmagnitudeasthe

signal produced by the plaque lipids. In accordance with the

differencesfoundbyvisualinspectionoftheIVPAimages,both

lipidmapsindicatelipidsinthetoppartofthelesion,aswellasin

theperi-adventitial tissueregion aroundthevesselwall.Inthe

1.7

mm

lipidmaphowever,moreintraplaquelipidsaredetected,

duetothehighersignalenhancementandthereforebettersignal

to noise ratio. The signal from the calciﬁcation is suppressed

successfullyinbothcases.TheenlargementsoftheOROlipidstain

(Fig.6eandf)oftheareaindicatedaslipidrichinthelipidmaps

revealthepresenceoflargerextracellularlipiddroplets,whereas

the lipids in all other parts of the lesion are intracellular or

containedinsmallextracellulardroplets.Thispreferential

detec-tionispossiblycausedbyahigherGru¨neisencoefﬁcient,ahigher

Fig.4.Lipiddetectioninalipid-containingvesselphantomusingsIVPAat1.2and1.7mm.(a)1205nmand(b)1235nmcombinedIVPA/IVUSimages(IVPA50dB,IVUS65dB) ofPVAphantomﬁlledwithcholesterol(bottom),cholesterololeate(right),cholesterollinoleate(top)andperi-adventitialtissue(left).(c)Lipidmapbasedon2-wavelength relativedifferencebetweenthePAsignalat1205nmand1235nm.(d).Co-registered1710nmand(e)1680nmcombinedIVPA/IVUSimages(IVPA50dB,IVUS65dB)ofthe samecrosssectionofthevesselphantom.(f)Lipidmapresultingfromthe2-wavelengthrelativedifferencebetweenthePAsignalat1710nmand1680nm.Bothlipidmaps areshownoverlaidonthecorrespondingIVUSimage.

0 0.2 0.4 0.6 0.8 1 c _cl _co _pl elastin collagen (PA 1205 −PA 1235 )/PA 1205 0.37 0 0.2 0.4 0.6 0.8 1 c _cl _co _pl elastin collagen (PA 1710 −PA 1680 )/PA 1710 0.23

(b)

(a)

Fig.5.RelativedifferenceofPAsignalathighandlowlipidabsorptionwavelengthsofindividual(unaveraged)IVPAspectrameasuredinthelipidcontainingphantom.(a)5th to95thpercentileoftherelativedifferenceofthe1205nmand1235nmPAsignalstrengthofcholesterol,cholesterololeate,cholesterollinoleateandperi-adventitiallipids (32,128,160and96spectra,respectively).Dataforelastinandcollagenareobtainedfrom[25].(b)5thto95thpercentileoftherelativedifferenceofthe1205nmand 1235nmPAsignalstrengthofsamelipidcomponents(samenumberofspectra).Elastindataobtainedfrom[27];collagendatafrom[28].c,cholesterol;cl,cholesterol linoleate;co,cholesterololeate;pl,peri-adventitiallipids.

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concentrationofthelipidsorabettermatchingofthegeneratedPA

signalfrequencytothebandwidthofthetransducer.

The datapresented here (inFigs. 3, 4and 6)showthat the

maximumlipidimagingdepthat1.2

mm

isapproximatelytwiceas

largecomparedto1.7

mm.

Theexactnumericalvaluedependson

theoptical(scattering andabsorption)andacoustic

(frequency-dependentattenuation)propertiesofthetissue.At1.2

mm,

lipid

signalwasreceivedfromtissuelayersinthearterywallatadepth

of5mm.

4. Discussionandsummary

This study assesses the lipid detection and distinction

capabilitiesofspectroscopicintravascularphotoacousticimaging

intwoabsorptionbands,around1.2

mm

and1.7

mm.

Weacquired

co-registeredsIVPA/IVUSdataofalipidcontainingvesselphantom

at6 wavelengthsin eitherspectral window.Correlation witha

cholesterolPAspectrum,asreferenceforatheroscleroticlipids,and

with a peri-adventitial tissue reference spectrum, using 6

wavelengths from 1185 to 1235nm, distinguishes very well

between atherosclerotic lipids and peri-adventitial lipids. The

same 6-wavelength correlation method applied on sIVPA data

from 1680 to 1751nm resulted in a poorer separation of in

particularcholesterololeateandperi-adventitialtissue.Applyinga

2-wavelengthrelativedifferencemethod,wesuccessfullydetected

allfourlipidcompoundspresentinthevesselphantominboth

wavelengthranges,withasuperiordetectionofallfourlipidsin

the1.7

mm

region.InanexvivosIVPA/IVUSmeasurementofan

humancoronaryartery,wefoundsuperiorplaquelipidcontrastin

the1.7

mm

wavelengthrange,withalowerpulseenergy(0.4mJ

versus1.2mJ at1.2

mm)

and sufﬁcient imaging depth (forthis

particularvesselcrosssection).Lowpulseenergyisanadvantage

becauseitlowerstheopticalpowerthatneedstobedissipatedin

vivo,andalsoreducesartifactsinIVPAimaging causedbylight

absorptioninthecatheter.

Therelativedifference betweentwowavelengths isa robust

parametertodetectthelipids,alsointhepresenceofstrongwater

absorptioninthelonger wavelengthband, aslongasthesignal

received from the lipids is above the noise level. Note that a

minimumoftwowavelengthsisrequiredinboththe1.2

mm

and

the 1.7

mm

wavelength range to distinguish lipids from other

vesselwallconstituents;theIVPAsignalgeneratedbycalciumisin

theorderofmagnitudeofthesignalfromlipidsaround1.7

mm

and

evenhigheraround1.2

mm.

Minimization of the required number of wavelengths is

important for clinical application: the acquisition speed is

inversely proportional tothenumber of PA acquisition needed

tocomposeanimageline.Lightsourcesareexpectedtorepresenta

signiﬁcant fraction of the cost of an IVPA system for clinical

applicability, presentinganotherreason tolimit the number of

Fig.6.LipiddetectioninanatherosclerotichumancoronaryarteryusingsIVPAat1.2and1.7mm.(a)1205nmand(b)1235nmcombinedIVPA/IVUSimages(IVPA25dB, IVUS40dB).(c)Lipidmapbasedon2-wavelengthrelativedifferencebetweenthePAsignalat1205nmand1235nm.(d).1710nmand(e)1680nmcombinedIVPA/IVUS images(IVPA25dB,IVUS40dB).(f)Lipidmapresultingfromthe2-wavelengthrelativedifferencebetweenthePAsignalat1710nmand1680nm.Bothlipidmapsareshown overlaidonthecorrespondingIVUSimage.(g)Lipidhistologystain(ORO);lipidsarestainedred;calcificationisstainedblack(h)5magnificationofthepartofthe atheroscleroticplaqueindicatedaslipidrichbythelipidstains(areaoutlinedinblackin(g)),showslargerextracellularlipiddroplets,whilethelipidsinallotherpartsofthe lesionareintracellularorcontainedinsmallextracellulardroplets.(i)4magnificationofareaoutlinedinblackin(h).

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wavelengths. We demonstrated here that two wavelengths are

sufﬁcient,in principle, todetectlipids, but notto differentiate.

Noisereductionintheacquisitionandmoresophisticatedanalysis,

targetedtoexploitspectraldifferencesbetweenthevariouslipid

tissues,mayyieldmoreinsightfromatwo-wavelength

combina-tion.

Thisstudyis qualitativeindesign, exploringseveralanalysis

methodsappliedtothedifferentlipidabsorptionbandsforlipid

detectionanddifferentiationbysIVPA.Alargerquantitativestudy

willbeperformedinthefuturetodeterminethemostfavorable

wavelengthsandtheappropriateparametersusedforprocessing.

Suchastudycouldalsoestablishwhethertheimagingdepthat

1.7

mm

issufﬁcient toimagethevesselwallof largercoronary

arteries,suchastheleftmainstem,completely.Alargerexvivo

studywillalsoelucidatetherepresentativenessofpure

cholester-ol,cholesterololeate,andcholesterollinoleatefortheabsorption

spectra of real atherosclerosis. It will provide insight into the

naturalvariabilityoftheabsorptionspectrum,whichtheeventual

choiceofwavelengthcombinationswillhavetotakeintoaccount.

Photoacoustic imaging seems to favor the detection of larger

extracellularlipiddroplets.ToquantifythesensitivityofsIVPAto

thedifferent forms and sizesof intraplaque lipids, a statistical

analysisofalargerdatasetshouldbeperformed.

In summary, we presented the lipid detection and typing

capabilitiesof1.2

mm

and1.7

mm

sIVPA.We observedsuperior

lipid differentiationin theshorter wavelengthrange in a lipid

containing vessel phantom. In the longer wavelength range,

however,intraplaquelipiddetectionwasimproved,bothin the

vesselphantomaswellasinanatherosclerotichumancoronary

artery,withlowerpulseenergy.

Conﬂictofintereststatement

Theauthorsdeclarethattherearenoconﬂictsofinterest.The

fundingagencyhadnoinvolvementinthestudy,thewritingofthe

manuscript,oritssubmission.

Acknowledgement

Thisworkwasfunded by theDutchTechnologyFoundation

(STW)throughthe2007SimonStevinMeestergrant(STW10040).

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KristaJansenreceivedherM.S.inTechnological Devel-opmentStudiesfromtheEindhovenUniversityof Tech-nologyandherM.S.inPhysicsfromtheUniversityof NorthTexas, where shestudied thenear-threshold positronimpactionizationofhydrogen.HerPhD re-searchwiththeDepartmentofBiomedicalEngineering oftheErasmusMedicalCenteronintravascular photo-acousticsisfocusedonthedetectionand characteriza-tion of human atherosclerotic plaques using spectroscopicphotoacousticimaging.Sheiscurrently workingasamedicalphysicistintrainingatAudiology CenteroftheVUUniversityMedicalCenterAmsterdam.

MinWuwasborninFujian,China.ShereceivedtheB.Sc. inelectronicinformationengineeringfromtheWuhan University,Wuhan,China,in2006,andthePhDdegree incommunicationandinformationsystemsfromthe sameuniversityin2011.MissWuiscurrentlypursuing adoctoratedegreeinIntravascularphotoacousticsat ErasmusMedicalCenter,Rotterdam,TheNetherlands, underthe guidance of Dr.Gijs vanSoest and Prof. AntoniusF.W. vanderSteen.Herresearchinterests includeIntravascularphotoacousticsandphotoacoustic imagingandspectroscopy.

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