Quantitative photoacoustic integrating sphere (QPAIS) platform for absorption coefficient and Gruneisen parameter measurements: Demonstration with human blood

Quantitative

photoacoustic

integrating

sphere

(QPAIS)

platform

for

absorption

coefficient

and

Gru¨neisen

parameter

measurements:

Demonstration

with

human

blood

Yolanda

Villanueva-Palero,

Erwin

Hondebrink,

Wilma

Petersen,

Wiendelt

Steenbergen

*

BiomedicalPhotonicImagingGroup,MIRAInstituteforBiomedicalTechnologyandTechnicalMedicine,UniversityofTwente,POBox217,7500AEEnschede, TheNetherlands

1. Introduction

Quantitativephotoacousticimaging(QPAI)inbiomedicineaims

at determining target chromophore concentrations such as

endogenoushemoglobinin humanbloodorexogenous contrast

agentlevels[1].Accuratemeasurementofconcentrationscanbe

obtainedfromtheabsorptioncoefficient

m

aofabsorbersofknown

molarextinctioncoefficients.InPAimages,

m

acanbeaccurately

measured if the Gru¨neisenparameter

G

of thetarget

chromo-phoresisknown [1]. Currentphotoacousticimaging techniques

estimatetheinitialpressuredistribution

s

owhichisaproductof

thesefactorsandthefluencedistribution

F

:

s

o=

Gm

a

F

[1].

Accu-ratemeasurementofeachfactorcangiveagoodestimationof

s

o

whichcanleadtocontrastonphotoacousticimageswhichhasa

quantitative interpretation. Reconstruction algorithms usually

assumeaconstant

G

foralltargetabsorberswhereinmeasurement

of

s

o mainly indicates the absorbed optical energy density

m

a

F

.However,differentmaterialshavedifferent

G

valuessince

G

is also directly related to the material’s thermodynamic

propertiessuchasthermalexpansioncoefficient

b

,specificheat

Cp,andspeedofsound

v

s,asitisdefinedas

G



bv

2s=Cp[2,3].For

example,inbiologicaltissues,

G

variesfromaround0.14forblood

[4]to0.80forfat[5].Severalpublicationshavereporteddifferent

techniquestomeasure

G

ofbiologicalchromophores[4–6].

How-ever,theexperimentalsetupsrequireabsolutedetectionsensitivity

measurements of the optical and acoustic signals and involve

stringentalignmentbetweentheincidentlightandtargetabsorber

and acoustic detector which may not be very convenient for

measuringwithliquidsamples.Inthispaper,wepresentamethod

forsimultaneouslymeasuring

m

aand

G

ofsmallvolumesofabsorbing

andscatteringliquidsinjectedina softtransparenttubemounted

through two integrating spheres [7]. With integratingspheres as

platform,uniformilluminationon thetargetabsorberisachieved.

Measuring

m

aofabsorbingsamplesinatubeinsideanintegrating

sphereispossibleeveninthepresenceofscattering.Thismethodof

determining

m

aiscombinedwiththetechniqueformeasuring

G

of

target absorbers in photoacoustic setup. The coupled integrating

sphere system is referred to as the quantitative photoacoustic

integratingsphere(QPAIS).Anequationformeasuring

m

ausingthe

integratingsphereisderived.Detailsoftheexperimentalsetupand

proceduresareenumerated.Absolutemagnitudesofopticalenergy

andpressurearenotnecessaryfordetermining

m

aand

G

;insteadan

insitucalibrationofthesystemisdonepriortomeasurementwith

samples ofinterest. The use ofthe system is demonstratedwith

measurementsonhumanbloodsamples.Measurementsaredoneat

roomandbodytemperaturesusinganincubator.

ARTICLE INFO Articlehistory:

Received31March2016

Receivedinrevisedform24February2017 Accepted18March2017 Keywords: Photoacoustics Absorptioncoefficient Gru¨neisenparameter Integratingsphere Quantitativephotoacoustics Humanblood ABSTRACT

Quantitative photoacoustic imaging in biomedicine relies on accurate measurements of relevant materialpropertiesoftargetabsorbers.Here,wepresentamethodforsimultaneousmeasurementsof theabsorptioncoefficientandGru¨neisenparameterofsmallvolumeofliquidscatteringandabsorbing mediausingacoupled-integratingspheresystemwhichwerefertoasquantitativephotoacousticintegrating sphere(QPAIS)platform.Thederivedequationsdonotrequireabsolutemagnitudesofopticalenergyand pressurevalues,onlycalibrationofthesetupusingaqueousinkdilutionsisnecessary.Asademonstration, measurementswithbloodsamplesfromvarioushumandonorsaredoneatroomandbodytemperatures usinganincubator.Measuredabsorptioncoefficientvaluesareconsistentwithknownoxygensaturation dependence ofblood absorption at750nm, whereas measured Gru¨neisenparameter valuesindicate variabilityamongfivedifferentdonors.AnincreasingGru¨neisenparametervaluewithbothhematocritand temperatureisobserved.Theseobservationsareconsistentwiththosereportedinliterature.

ß2017UniversityofTwente.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

* Correspondingauthor.

E-mailaddress:w.steenbergen@utwente.nl(W.Steenbergen).

ContentslistsavailableatScienceDirect

Photoacoustics

j ou rna l h ome p a ge : w ww . e l se v i e r. co m/ l oc a te / p a cs

http://dx.doi.org/10.1016/j.pacs.2017.03.004

2213-5979/ß2017UniversityofTwente.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

2. Methodology

2.1. Doubleintegratingspheremethodandexperimentalsetup

The methodfor measuring

G

of absorbing liquids using an

integrating sphere was already published [8] and was also

implemented and briefly described in this paper. The main

modificationswereas follows: (1) thecentralfrequency of the

transducerusedforphotoacousticdetectionwas5MHz,(2)asoft

transparentpolyethylenetubewithinnerdiameter=0.58mmand

outer diameter=0.96mm was used and (3) another similar

integrating sphere with the same physical properties was

connectedtothe one usedfor photoacoustic measurements so

thattheabsorptioncoefficientoftheabsorbingliquidinsidethe

tubecouldbesimultaneouslymeasuredusingspectrophotometry.

Fig.1showsaschematictopviewillustrationofthecoupled

integratingspheresetups.Forclarity,onlytheopticalsourcesand

detectorsareshown.Thetransducer(OlympusPanametricsNDT

V309 5MHZ/0.5in. 878182), positioned along the z-axis, the

amplifier(PanametricsNDTUltrasonicPreamp5678)and

oscillo-scope (200MHz, 2 GS/s, Tektronix TDS 2022C/24C) used for

photoacousticdetectionarenotshownontheillustration.Details

onphotoacousticmeasurementsaregivenin[8].

Asoftpolyethylenetube(Portex,SmithsMedicalInternational,

Ltd.,UK)withinnerdiameterof0.58mmwasinsertedthrough

smallholesoneachintegratingsphere(ThorlabsIS200)suchthat

the tube was positioned horizontally inside both spheres. The

verticalheightofthetube wasabout4mm aboveeach sphere

centerwhichensuredthatlightwasdirectlyincidentonthesphere

wall,avoidingdirectilluminationofthesample.

Absorptionmeasurementwasdoneusinganair-filled

integrat-ingsphere1.Ahalogenlamp(AvantesAvaLight-Hal)lightsource

wasfiber-coupledtothisintegratingspherewhileaspectrometer

(AvantesAvaSpec2048)connectedtoanotherportonthissphere

usingasimilarfiberopticwasusedtomonitorthevariationonthe

opticaloutputsignal.

Photoacousticmeasurementof

G

wasdoneusingintegrating

sphere2whichwasfilledwithwaterforacousticmatchingwith

the transducer. A pulsed laser source (OpoletteTM 532I) with

wavelengthof750nm,pulselength7ns anda pulserepetition

frequencyof20Hzwasconnectedtothissecondsphereusingan

optical fiber (Newport, 0.22 NA, core diameter of 1mm).

Photodetectors (Thorlabs DET10A/M-Si detector) were used to

monitortherelativeinputandoutputlightenergy.Theuniform

illuminationonthetubewasnotaffectedbytheintroductionof

water inside the sphere cavity since the sphere wall coating

(spectralonmaterial)maintainsitshighreflectancepropertyeven

inthepresenceofwater.Moreover,thetubewasinsertedinavery

smallhole(withdiametersameastheouterdiameterofthetube)

throughtheappropriateportplugssuchthatwaterdidnotleakout

ofthesphere.

For body temperature measurements, the two-integrating

sphere setup was placed inside an incubator where ambient

temperature adjustments and measurements were possible. A

built-inblowerwarmedtheairwhileathermalsensorcontrolled

the temperature inside the incubator. It took approximately

10min to increase from room temperature 228C to body

temperatureof378C.Thereservoirswiththemediumofinterest

werealsoplacedinsidetheincubatorsothattheywereinthermal

equilibrium with the entire system during optical and

photo-acousticmeasurements.Anotherthermalprobe(National

Instru-ments NI USB-TC01) was used to monitor and record the

temperaturemeasurements.Immediatelybefore absorptionand

photoacoustic measurements, the air temperature within the

incubator,water temperatureinside theintegrating sphereand

temperatureofthesamplesweremeasured toensurethatthey

wereallinthermalequilibrium.Thetemperaturewasmonitored

throughouttheexperiments,withthethermalprobepositioned

closetothetubethatgoesintointegratingsphere2.

2.2. Preparationofhumanbloodsamples

Bloodsamplesfromhealthy donors wereobtained fromthe

Experimental Center for Technical Medicine (ECTM) of the

UniversityofTwente(UT),whichimplementsproperethicsand

approved procedure in utilizing humans and humantissues in

research.Bloodwasdirectlydrawnintovacuumsealedtubeswith

anticoagulant (either EDTA or heparin) for temporary storage.

Immediatelybeforeabsorptionandphotoacousticmeasurements,

bloodfromeachvacuum-sealedtubewaspipettedintoEppendorf

tubestoobtainthreesamplesfromthesamedonor,eachabout

1mlinvolume.Thisstepensuredthattheoxygensaturationofthe

hemoglobininthebloodwouldnotabruptlychangewhenblood

wasinjectedintothetubeintheintegratingspheresetup.

Severalfreshhumanbloodsampleswerecollectedonvarious

days. Measurements were done to investigate the measurable

valuesof

m

aand

G

forbloodsamples(1)drawnfromthesame

donoronvariousdayswithnewsetupcalibration(2)drawnfrom

threedifferentdonorsonthesamedaywithsamesetupcalibration

and(3)drawnfromvariousdonorsonvariousdayswithnewsetup

calibration. Absorption and photoacoustic measurements were

donewithinonetotwohoursafterthebloodsamplewasdrawn

fromthe donor.Measurements on thesame dayweredone to

indicateusingthesamesetofcalibrationconstantsformeasuring

m

aand

G

ofbloodsamplesfromvariousdonors.Ontheotherhand,

measurements on various days implied investigation on the

measurablevalues using new calibrationmeasurements of the

systemsincecalibrationwasalwaysdone immediatelypriorto

measurementswiththesamplesofinterest.

Forhematocritdependenceinvestigation,wholebloodsamples

inside the vacuumsealed tube wereplaced in a centrifuge for

about10minat2000rpmuntilallRBCsettledintothebottomof

thetube.PlasmaandRBCwereseparatedandwerepipettedinto

Eppendorf tubes to obtainabout threesamples fromthe same

donor,eachabout1mlinvolume.Mixturesofredbloodcells(RBC)

andplasmainvaryingRBCconcentrations(forexample,30vol%

RBC tohave hematocrit of approximately 30%) wereprepared.

Actual hematocrit values were determined by measuring the

relativeheightoftheRBCcolumnincapillarytubes.

In order to have absorbing plasma samples at the 750nm

wavelength,smallamountofindocyaninegreendyesolutions(less

Fig.1.Top-viewschematicdiagramofthedoubleintegratingspheresetupfor measuringabsorption coefficientmaand Gru¨neisenparameterG. Thetubeis

positionedapproximately4mmabovethecenterplanewheretheopticalportsare located.

than10vol%)wereaddedtoplasmasamplespriortopipettinginto

Eppendorf tubes to obtain three 1ml samples from the same

donor,whichcorrespondstobloodssampleswithzerohematocrit.

2.3.

m

ameasurement

Eachabsorbingsampleofinterestwasinjectedintothetube

untilitflowedouttheotherendtoensurethatthesamesample

wasmountedinbothintegratingspheres.Simultaneousdetection

oftheopticaland photoacousticsignals weredonein a similar

mannerasinthecalibrationmeasurement.

Theabsorptioncoefficient

m

aoftheabsorbingbloodsample

insidethetubemountedintheintegratingsphere1wasderived

usingsimpleenergybalancewithinthesphere.Theincidentlight

energyEinwasdistributedoverandwasabsorbedbythevarious

parts within the sphere, such that Ew, Ea and Eout are the

magnitudesoftheabsorbedenergybythespherewall,absorber

tubeandoutputport,respectively.Fromsimpleenergy balance

considerations,anequationcouldbewrittenasfollows

Ein¼EwþEaþEout (1)

Eq.(1)couldalsobewrittenintermsoftheuniformfluence

F

withinthesphere

Ein¼cw

f

þca

f

þco

f

(2)

cw,caandcodependonthematerialsused.Forthecaseofaweakly

absorbing sample inside the tube (relatively low absorption

coefficient which is less than 2mm1_{), c}

a=

m

a V, such that

absorptionwasuniformovertheentirephysicalvolumeV.Using

Eout=co

F

,Eq.(2)becomes

Ein¼cw Eout co þ

m

a VEout co þ Eout (3) Eq.(3)simplifiesto Ein Eout ¼aþb

m

a (4) wherea¼ðcw=coÞþ1andb=V/co

Moreover, Ein could bedetermined using Eout if there is no

absorbingsampleinsidethetubesinceEin=cinEout,noabsorber.Also,

Eout¼coutE0out where cout is a constant that takes into account

experimentalfactorssuchassensitivityofdetectionand

conver-sionfromabsolutevaluetoarbitraryunits,forexampleJoulesto

Counts,as wellas theattenuation due tofiber coupling.Thus,

Eq.(4)couldbewrittenas

E0in E0 out ¼AþB

m

a (5) whereE0 in¼E 0

out;noabsorber,themeasuredoutputsignalwhenthere

isnoabsorberinsidethetube,andAandBareconstantswhich

couldbemeasuredexperimentallyviaacalibrationprocedureas

describedabove.RearrangingEq.(5)gives

m

a¼ ðE0in=E 0 outÞA B (6) 2.4.

G

measurement

ThecorrespondingGru¨neisenparameter

G

wasmeasuredusing

thefollowingequation[8]

G

¼Vppðcþ

m

aVÞ E0in;PAk

m

a

(7)

Here, Vpp was the voltage-peak-to-peak amplitude of the

detectedphotoacousticsignalgeneratedbytheabsorbingblood

samplewith

m

aandvolumeV.

m

awasdeterminedvia

simulta-neousspectrophotometryusingintegratingsphere1asdescribed

above,whereasV=0.0134cm3_{,thephysicalvolumeofthetube.c}

andkaretheinstrumentconstantsdeterminedviathecalibration

methoddescribedinreference[8].E0in;PAistherelativeenergyof

the incident pulse measured by the photodetector. A detailed

derivationofEq.(7)isgiveninRef.[8].

2.5. Calibrationmeasurement

Acalibrationprocedurewasdonetodeterminetheinstrument

constants. Aqueous ink dilutions were used as absorbers with

known

m

a values as measured by the standard transmission

spectrophotometry technique(Shimadzu UV-VIS). BlackEcoline

ink(RoyalTalensEcoline7008265)wasdilutedwithdeionized

anddemineralizedwaterinordertomakeatleastfive

concentra-tionswith

m

avaluesrangingfromabout0.2to2mm1.Eachink

dilutionwasinjectedintothetubeuntilitflowedouttheotherend

toensurethatthesameabsorbingsamplewasmountedinboth

integrating spheres. Simultaneous measurements ofthe optical

outputandphotoacousticsignalsweredonebysynchronizingdata

collection via computer interface using AvaSpec and LabVIEW

software. The AvaSpec software recorded the amount of light

reachingtheoutputportofthespherewhichwasfiber-coupledto

the spectrometer that scans a spectrum between 400nm and

900nm wavelengths. With the assumption that only the

introductionoftheabsorbinginkwaschangedintheintegrating

spheresystem,therelativeinputlightenergyE0in;abswastakenas

the detected output signalwith only water (no absorbingink)

insidethetubewhereastherelativeoutputlightenergyE0

out;abswas

equivalenttothesignalwiththeabsorbinginkinsidethetube.

Absorptionotherthanthatduetoinkabsorptionwasassumedto

bethesameinallmeasurementsandcanbecancelledoutinthe

calculation for

m

a of the absorber in the tube. The necessary

constantsformeasuring

m

aofsampleswereobtainedfromaplotof

E0

in;abs=E0out;abs ratio versus

m

a values. On the other hand, the

LabVIEW program recorded the temporal photodetector and

transducer photoacoustic signals. The area under the curve of

thephotodetectorsignalwastakenastherelativeinputenergy

E0

in;PA,whereasthevoltage-peak-to-peakVppofthephotoacoustic

signal was proportional to the initially generated pressure

amplitude. To obtain the necessary instrument constants for

determining

G

,themeasuredVpp=E0in;PAratiowasplottedagainst

corresponding

m

avaluesofinkdilutions.Themeasured

m

avalues

oftheaqueousinkabsorberswereusedinE0

in=E0outversus

m

aand

Vpp=E0in;PAversus

m

acalibrationplotsasshowninFigs.2and3.

3. Results

Examplesofdetectedphotoacousticsignalswithinkabsorbers

andhumanbloodsamplesareshowninFig.2.Ascanbeseenin

Fig.2a,theVppamplitudeofthesignalincreaseswith

m

aofthe

aqueous ink calibration samples. The corresponding Vpp=E0in;PA

versus

m

aplotisgiveninFig.3b.Fromthisplot,thecalibration

constants are k=1.17m3_s1 _{and c=3.55}

105_m2_.

Simulta-neouswithdetectingphotoacousticsignals,thespectrumofthe

opticaloutputsignalfromthesecondsphereisalsomeasuredand

thecorrespondingE0

in=E0outratioforincreasing

m

aisgiveninFig.3a.

A linear fit on this plot gives A=1.02 and B=0.21mm1_.

Immediately afterobtainingthe calibrationdata, photoacoustic

measurementswithblood samplesinsidethetubearedone for

severaltimes.Exampleofthedetectedsignalsfromwholeblood

samplesisgiveninFig.2b.Eachplotcorrespondstotheaverageof

fivemeasurementsaveragedfrom128pulses.Asareference,the

detectedsignalwithwaterinsidethetubeisalsogiveninFig.2b

The corresponding E0in=E

0

calibrationconstantsAandBareusedinEq.(7)todeterminethe

m

a,bloodofthebloodsample.Thismeasured

m

a,blood,togetherwith

theconstantskandc,V=0.0134cm3_{andcorrespondingV}

pp=E0in;PA

aresubstitutedintoEq.(1)tocompute

G

blood.

3.1. Measurementswithfreshbloodsamplesfromthesamedonor

withcorrespondingsetupcalibrationonvariousdays

Absorptionandphotoacousticmeasurementswithfreshblood

samplesfromthesamedonoraredoneatvariousdays,usinganew

calibrationofthesetupeachday.Simultaneouswithabsorption

andphotoacousticmeasurements,oxygensaturation(SO2)values

of each blood sample are measured using anoximeter

(Avoxi-meter).ThevaluesgiveninTable1indicatethatmeasurablevalues

of

m

a,bloodoffreshbloodsamplesfromthesamedonorcanchange

atvariousdayswhich mainlydependontheoxygen saturation

(S02) levels. For the highest measured S02=93%,

m

a,blood=0.4310.009mm1, whereas for the S02=46%,

m

a,blood=0.8070.010mm1,whichisconsistentwiththegenerally

reportedvalueat750nm[9].Ontheotherhand,themeasured

G

blood

ranges from 0.16 to 0.18 with an average value of

G

blood=0.1660.008. The standard deviation is only 5% of the

averagevaluewhichindicatesthatthemeasureable

G

valueforblood

samplesfromthesamedonorisrepeatable.Moreover,themeasured

valueof0.166forthishumanbloodsampleisonly4%differentfrom

thereportedvalueofbovineblood[5].

3.2. Measurementswithfreshbloodsamplesfromdifferentdonors

withonesetupcalibration

Table2showsasummaryofthemeasured

m

a,bloodand

G

blood

from three different donors (labeled A, B, C) using one set of

calibrationplots.Threemeasurementswiththesamewholeblood

samplefromthesamedonoraredonewhichgivesthestandard

deviationvalues. The corresponding hematocrit foreach whole

bloodsampleismeasuredandisalsogiveninTable2.Measured

SO2valuerangesfrom46%to84%.ThelowSO2levelindicatesthat

thebloodsampleisindeedvacuum-sealedandminimallyexposed

toairfromthetimeitisdrawnfromthehealthyhumandonoruntil

it is placed intothe oximeter.Moreover, themeasured

m

a,blood

varies withthe measured SO2 values. In particular,as the SO2

increases,themeasured

m

a,blooddecreaseswhichisconsistentwith

thereportedbehaviorof

m

aat750nmwavelength[9].

Infurtherinvestigatingthedependenceof

G

bloodonhematocrit,

Fig.4showsaplotofthemeasured

G

bloodversushematocrit.For

zerohematocrit,measured

G

hct=0=0.1200.002whichiswithin

10%differentfromthereportedvalueforbovineserum[5].Asshown

inFig.4,themeasuredvalueof

G

increasedto0.1960.010for100%

hematocrit,orthecasewhenthereareonlyerythrocytesinsidethe

tube.The generaltrendisincreasing

G

forincreasinghematocrit,

except for between47 and60 which showsa slightdecrease in

G

.Moreover,measurementsaredonewithonlytheredbloodcells

(RBC)ofthesamplesdrawnfromthesamedonorsA,BandC.Table3

showsthemeasuredvaluesof

m

a,RBCand

G

RBCwhicharerelatively

higherthanthosegiveninTable2.Therelativelyhighervaluesof

m

a,RBCcanbeattributedtotheabsenceofplasmawhichismostly

(about 90%) water and has relatively low absorption at 750nm

wavelength whereas the higher

G

RBC values can be due to the

Fig.3.Exampleofcalibration plotsfor determiningtheconstants (a)A=1.02and B=0.21mm1 _(R2_{=0.99)usedforcalculatingm}

aand(b)k=1.17m3s1 and

c=3.55105_m2

(R2

=0.98)formeasuringG.

Fig.2.Exampleofdetectedphotoacousticsignalswith(a)aqueousinkdilutionsofknownmaand(b)humanwholebloodsampleinsidethetube(measuredthreetimes).

Measurementwithwater(blackline)isshownforreference.

Table1

MeasuredvaluesofabsorptioncoefficientmaandGru¨neisenparameterGforeachof

the bloodsamples from the same donor. Theabsorption and photoacoustic measurementsaredoneondifferentdaysandcorrespondingly withdifferent calibration ofthe setup.The average G valuefrom thesethree independent measurementsisGblood=0.1660.008.

Measurement ma,mm1(meanSD) G(meanSD)

1 0.8110.093 0.1690.008

2 0.4310.009 0.1570.003

modifiedthermophysicalpropertiesofanensembleofRBCcompared

tothatofwholeblood.Forexample,thethermalexpansioncoefficient

(

b

) ofRBCisrelativelyhigherthan thatofwholeblood,whichis

directlyproportionalto

G

.

3.3. Measurementswithfreshbloodsamplesfromvariousdonors

withcorrespondingsetupcalibrationonvariousdays

Theresultsofthemeasurementsfromvariousdonorsdoneon

variousdaysaresummarizedinTable4.Therangeofvaluesof

measured

G

forfreshhumanbloodsamplesfromfivedonorsvaries

from0.141to0.177, witha mean valueof 0.162and standard

deviation(SD)of0.016whichis10%ofthemean.Itshouldalsobe

notedthatthelowestmeasuredvalueiswithin20%differentfrom

thehighestmeasuredvalue,whichcanindicateaperson-to-person

variabilityin

G

thatmaybeattributedtovariationsinhematocrit

andbloodcompositionandcondition.

3.4. Comparisonbetweenmeasurementsatroomandatbody

temperatures

Table5shows

m

aand

G

valuesofbloodsamplesmeasuredat

room(228C)andbody(378C)temperatures.Opticalabsorptionof

thesamplesdoesnotchangewithtemperature.Ontheotherhand,

photoacousticefficiency

G

increasedwithtemperature.Forthisset

ofmeasurements,

G

increasedbyabout70%,from0.1500.011at

228Cto0.2450.006at378C.Measurementsat378Caredonefive

times. Table 6 shows a summary of measured values for blood

samplesfromfive differentdonors.Asnotedabove, thevaluesof

measured

m

achangedueto theinherentoxygenationlevelofthe

bloodsamples.Ontheotherhand,theaverage

G

=0.2260.015at

378Chas7%standarddeviationwhichindicatesthatthemeasured

G

atbodytemperaturealsovariesminimallyrelativetomeanvalue,

althoughthelowestmeasuredvalueof0.208isapproximately18%

differentfromthehighestmeasuredvalueof0.245whichissimilarto

thedifferenceobservedat228C.Comparingtheaverage

G

measured

at378Cwiththeonemeasuredat228C,measured

G

increasedby

about 40% with increased temperature. The increased value of

measured

G

canbemainlyattributedtothecombinedincreaseinthe

relevantthermalexpansionpropertiesofthedifferentcomponentsof

blood,suchasred bloodcellsandplasmawhichis mostlywater

whichincreaseswithtemperature[3,10].Furthermore,itisrecently

reported that the photoacoustic signal from blood samples with

varying hematocrit increases with temperature which gives an

indicationonthetemperaturedependenceof

G

[11].

4. Discussion

Prior to performing measurementswith blood samples,the

influenceoflightscatteringtomeasureablevaluesofabsorption

coefficients and Gru¨neisen parameterof aqueousink solutions

with intralipid was investigated as presented in our paper

[12].Resultsshowthattherangeofvaluesthatcanbeaccurately

measuredusingourintegratingspheremethodare

m

a<1.5mm1

and

m

0

s<3 mm1,whicharewithintherangeofreportedvalues

formostbiologicalfluidsincludingblood,atinfraredwavelengths.

Photoacoustic measurementswiththeseabsorbingaqueousink

samples (with and without the intralipid) give values of the

Gru¨neisenparameterclosetothatofwater,asexpectedsincethe

Table2

Measuredvaluesofoxygensaturation(SO2),hematocrit(hct),absorptioncoefficientmaandGru¨neisenparameterGforeachofthebloodsamplesfromdonorsA,BandC.

Samples SO2,%(meanSD) Hematocrit,%(meanSD) ma,mm1(meanSD) G(meanSD)

A 634 492 0.6940.026 0.1770.005

B 844 461 0.5800.016 0.1660.006

C 463 471 0.8070.010 0.1730.002

Fig.4.MeasuredGru¨neisenparameterGversusbloodhematocrit.

Table3

Measuredvalues ofabsorptioncoefficientma andGru¨neisenparameterGfor

sampleswith100%hematocrit(containingonlyredbloodcells)fromeachofthe blood samples from donorsA, B and C. Three simultaneous absorption and photoacousticmeasurementsaredoneforeachsampletoobtainthestandard deviation(SD).

Samples ma,mm1(meanSD) G(meanSD)

A 0.9490.022 0.1890.006

B 0.8180.015 0.2070.019

C 0.9060.011 0.1920.009

Table4

MeasuredvaluesoftheGru¨neisenparameter

G for blood samples from five different donors(donorsD1–D5).Threesimultaneous absorptionandphotoacousticmeasurements are done for each sample to obtain the standarddeviation(SD). Samples G(meanSD) D1 0.1770.005 D2 0.1520.020 D3 0.1660.008 D4 0.1760.007 D5 0.1410.003 Table5

maandGvaluesofbloodsamplesfromonedonormeasuredatroom(228C)and

body(378C)temperatures.

Temperature(8C) ma(mm1) G

22 0.5010.028 0.1500.011

37 0.4930.034 0.2450.006

Table6

maandGvaluesofbloodsamplesfromfivedifferentdonorsmeasuredatbody

temperatureof378C. Samplenumber ma(mm1) G 1 0.4930.034 0.2450.006 2 0.7380.003 0.2160.005 3 0.4970.012 0.2080.007 4 0.9080.032 0.2260.005 5 0.9050.012 0.2400.005

samplesarecomposedmostlyofwater.Itshouldbenotedthatthe

absorptioncoefficientcanalsobe measured fromthetemporal

profileofthephotoacousticsignalusingbackwardmodedetection

[13,14].Thisphotoacousticmeasurementoftheopticalabsorption

is also investigated for turbid medium using Monte Carlo

simulations [15]. In this research, it hasbeen shown that the

diameteroftheincidentlaserbeamcanbechosensuchthatthe

absorbed optical energy, which is proportional to the

photo-acoustic amplitude, is linearly dependent on the absorption

coefficient, independent of the scattering coefficient. In our

integratingspheremethod, theincidentlight ishomogeneously

distributed on the absorbing target independent of the beam

diameterfor

m

a<1.5mm1and

m

0s<3 mm1.

Absorption and photoacoustic measurements were done on

fresh blood samples from healthy human donors. No further

analysesonthesamplesweredonetocheckforsimilaritiesand

differencesintheir physiologicalconditions.Therefore,different

measurement scenarios were designed and implemented to

investigatetheinfluenceofvariationsineitherthebloodsamples

ortheexperimentalsetup.Performingmeasurementsonvarious

days(notnecessarilyatregularintervals)wereaimedatinvestigating

onthemeasurablevaluesof

G

withvariousfreshbloodsamplesusing

theintegratingspheresetupwithnewcalibrationmeasurementfor

everysample.Thiswastoinvestigatethestabilityofthemethodand

setupandhowanyperturbationonthesystem,suchaschangingthe

absorbingsampleordoingnewinstrumentcalibration,couldaffect

the measurable values. Results indicated that the measured

m

a

changes with blood oxygenation and the measured

G

of blood

samplesfromaparticulardonorwasthesameforthreeindependent

measurementswitharelativeerrorofonly5%.

To investigatefurther,measurementswithmorefreshblood

samplesfromvarioushumandonorsaspiratedondifferentdays

wereperformed.Resultsindicatedthatthevaluesofmeasured

G

varyamongfivedifferentdonorswithamaximumdifferenceofup

to20%,whichistwicethemeasuredexperimentalerrorobtained

withcalibrationofthesystem,asdescribedin[8].Thisobservation

suggestedthatthe

G

of humanblood mayvary fromperson to

personwhichdependsonphysiologicalfactors.Unfortunately,no

furtheranalysisonthecompositionandconditionoftheobtained

bloodsampleswasdone.Onepossiblereasonforthedifference

couldbethevariationsinthehematocritofthesamples.However,

based on the results of hematocrit dependence measurements

showninFig.4,the

G

valuesof0.14and0.17wouldcorrespondto

hematocrits of 20 and 60, respectively, which are beyond the

normalrangeforhealthywholeblood.Anotherpossiblesourcefor

thevariationinmeasured

G

couldbethevariationinthesample

temperature.Fromthemeasuredaveragevalues,anincreasefrom

0.16at228Cto0.22at378Cimpliesthat0.004increasein

G

per

degree increase in temperature. This would give a temperature

fluctuationof10degreesbetween

G

=0.14and0.17,whichisvery

unlikelysincethemeasuredambienttemperatureduringtheentire

experimentvarieswithin onedegreeonly.Therefore,variationsin

hematocritandtemperature,althoughtheymayhaveasmalleffect,

werenotthemainreasonfortheobservedvariationin

G

ofblood

fromvarioushuman donors.Moremeasurementscan bedoneto

exploreonthevariabilityof

G

fromperson-to-personandtofurther

investigatetherelevantphysiologicalfactorsaffectingthis.

ResultsshowninFig.4indicatedthatthemeasuredvalueof

G

generallyincreaseswithbloodhematocrit.Bloodsamplewithzero

hematocritcorrespondstosamplewhich containsbloodplasma

and that with 100% hematocrit corresponds to sample which

containsonlyredbloodcells.Thedifferenceinthevaluesof

G

for

these two samples (with zero and 100% hematocrit)could be

mainly attributed to the difference in the composition (with

different thermophysical properties). For the samples with

hematocritbetween zeroand100%, thecombinationof plasma

andredbloodcellsmayhavedifferentthermophysicalproperties

whichgiveadifferenteffective

G

values,moreredbloodcellsmay

indicatehigher

G

.Ithasalsobeenreported(asdescribedinRef.

[16]) that scattering effects could result to higher value of

calculated

G

.

Theobservedvariationin

G

withhematocritandtemperature

could have consequences for in vivo photoacoustic imaging of

microcirculationofblood.Smallerbloodvesselstendtohavelower

hematocrit, the so-called Fahraeus effect, which lowers the

effectivebloodviscosityinthesmallervesselsofthe

microcircu-lation,especiallyinneonates[17,18].

The dependenceof

G

on blood hematocrit and temperature

couldformacomplicationforquantitativephotoacousticimaging,

with the aim to determine true absorption coefficients. For

example,basedon theobtained results,a changein hematocrit

values from50% to20%, whichisfoundinthemicrocirculation

whencomparingbloodinlargemicrocirculatoryvesselswithsmall

vessels,coulddecreasetheGru¨neisenparameterbyapproximately

20%.Furthermore,afive-degreevariationoftemperaturewithin

thebody couldinduceatleast 10%variations oftheGru¨neisen

parameter.Such afive-degreetemperaturedifference mayexist

betweenthebodycoreandtheskin,forinstance.Hence,natural

variations in local temperature or hematocrit may cause a

significant variation in the Gru¨neisen parameter. Since the

photoacousticstress

s

oisrelatedto

G

,

m

aandopticalfluence

F

as

s

o=

Gm

a

F

, thesenaturalvariationsin thevalueof

G

could

introduceanextrauncertaintyintotheproblemofquantifying

m

a,

ontopoftheuncertaintyinfluence

F

.

Itshouldalsobenotedthatinourmeasurementswithaqueous

ink and blood samples, the difference in acoustic impedance

betweencalibrationmediumandbloodmediumwasnotexplicitly

considered in the calculations. The polyethylene tube used to

mountthesampleinsidetheintegratingspheresystemwaschosen

becauseithasacousticimpedancesimilartothatofsoftbiological

tissues.Eventhoughtheacousticimpedanceisnotconsideredin

the calculations, the

G

values we obtained are in quite good

agreementwiththatforbovinebloodreportedinrelatedliterature.

Moreover,theobservationsforvaryinghematocritandtemperature

are notaffected by a potential difference of acoustic impedance

betweenbloodandthecalibrationmedium.Alsothevariationsof

G

betweensubjects,orbetweendays,arenotaffectedbythis.

5. Conclusion

Amethodandsystemfordeterminingtheabsorption

coeffi-cient

m

a and Gru¨neisen parameter

G

liquid absorbing and

scatteringsamplesweredesignedandimplementedusingcoupled

integratingspheres.Onespherewasusedasaplatformfordoing

absorptionmeasurementsandanothersphereforphotoacoustic

measurementswiththesampleinside atubemounted

simulta-neouslythroughbothspheres.Usingthemeasuredrelativeoptical

outputratios,alinearequationfordetermining

m

awasderived

basedonsimpleenergybalancewithinthesphere.Thismeasured

m

awasusedincalculatingfor

G

ofthesample.Theapplicationof

the developed platform referred as quantitative photoacoustic

integrating sphere (QPAIS)wasdemonstrated bymeasuring

m

a

and

G

ofhumanblood.Atroomtemperaturemeasurements,fora

particular donor, the measured

m

a of blood ranged from

0.807mm1 _{to 0.431mm}1 _{with S0}

2 values of 46% and 93%,

respectivelyand corresponding measured

G

=0.1660.008 was

repeatable for three independent measurements withnew setup

calibrationatvariousdays.Thisvalueisingoodagreementwiththe

valuesobtainedforbovineblood[5].Moremeasurementswithfresh

bloodsamplesfromvariousdonorsindicatedadecreasing

m

avalue

forincreasingbloodoxygenationlevelswhichisconsistentwiththat

blood samples fromfive human donors, withapproximately 20%

differencebetweenlowestvalue0.141andhighestvalue0.177,each

within15%differencefromthemean.Measurementswithvarying

bloodhematocritindicatedthat

G

increaseswithbloodhematocrit.

However,thevariationinwholebloodhematocritwastoosmallto

causetheobserved20%variationin

G

.Thebloodsamplesobtained

wereassumedtobefromhealthydonors.Theactualphysiological

stateofthebloodsamplesanditseffectonthemeasurable

G

couldbe

furtherinvestigated.Moreover,measurementsatbodytemperature

of 378C gave an average

G

=0.2260.015 which is about 40%

different from the measured value at 228C. This observation of

increasing

G

valuewithincreasingtemperaturewasconsistentwith

resultsinRef.[11].

The shown dependence of Gru¨neisen parameter on blood

hematocritandtemperaturecouldformanextracomplicationfor

quantitativephotoacoustics,becauseofthenatural variationsof

hematocrit found in the microcirculation, and temperature

differencesbetweenthebodycoreandtheskin.

Themethodpresentedherecouldbeusedformeasuringwith

other weakly absorbing liquid samples which are relevant to

biomedicine,particularly the target absorbers in photoacoustic

imaging.Itshouldbenotedthatthedemonstrationofthemethod

waspresentedhereusingonlyonewavelength.Inprinciple,QPAIS

could be used with a range of wavelengths such that further

quantitative investigations could be done. Additionally, the

requiredincidentenergy perpulsewasrelativelylow suchthat

thelasersourcecouldbechangedtoaportablelaserdiodewith

reasonablyshortpulseduration.Alightsourcewithlessenergyper

pulse(approximately<3mJperpulse)couldbeused,insteadof

thehighenergysourcesusedforphotoacousticimaging.

Conflictofinterest

Nonedeclared.

Acknowledgements

TheauthorsacknowledgeTheNetherlandsTechnology

Foun-dationSTWforthefinancialsupporttothisresearch(Vicigrant

10831).Likewise,theauthorssincerelyappreciate thekindand

generous assistance of the Experimental Center for Technical

Medicine(ECTM)oftheUniversityofTwente,togetherwiththe

varioustechniciansanddonors,whofacilitatedthesupplyoffresh

humanbloodsamplesusedintheabsorptionandphotoacoustic

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YolandaVillanueva-Paleroobtainedherbachelorof scienceinappliedphysicsdegreefromtheNational InstituteofPhysicsattheUniversityofthePhilippines in 2000. She did her bachelor thesis project on holographicopticaldataencryptioninaniron-doped lithiumniobatecrystalatthePhotonicsResearchgroup. InDecember2003,shealsojoinedasix-monthresearch trainingprogramattheformerPrecisionInstrument Development Center of Taiwan Republic of China, wheresheperformedpulsedlaserdepositionofzinc oxide thinfilms. She obtained a masterof physics degreefromtheVrijeUniversityAmsterdaminAugust 2010.HermasterthesiswasonCasimir-likeeffectin granularfluids.InJanuary2016,sheobtainedherPhD degreeattheUniversityofTwentewhereshedevelopedaquantitativephotoacoustic integratingsphereplatformformeasuringtheGru¨neisenparameter,opticalabsorption andfluorescencequantumyieldofbiomedicalfluids,underthesupervisionofProf. WiendeltSteenbergenofthebiomedicalphotonicimaginggroup.

ErwinHondebrink receivedhis bachelordegreein biomedicalelectrical engineering in 1998 at Hoge-schoolEnschede.From1999to2015,heworkedalmost continuouslyasaresearchengineerattheUniversityof Twente.HeworkedintheLowTemperaturegroupand the Biomedical Imaging group on various research projectsinmicrocooling,laserdoppler,LASCA, photo-acousticsandacoustooptics.Hisfocusisonsoftware development,dataacquisition,visionandelectronics. In2007,heworkedatPerimedABinSwedenonthe developmentofaLASCAsystem.From2009to2010,he workedatOstendumBVonaportablebiosensorforthe detectionofbacteria,viruses,yeasts,andbiomarkers. WilmaPetersenstudiedmedicallaboratoryeducation withthespecializationMedicalChemistryandClinical ChemistryattheHogeschoolEnschede.Shereceived bothdegreesin1998.ShestartedworkinginAcademic Medical Center (AMC) in Amsterdam as research analystwereshegeneratedproteinstodostructural functionanalysis.In2005,shejoinedtheBiomedical PhotonicImaginggroupattheUniversityofTwente, Enschedewhereshepreparedphotoacousticcontrast agentssuchasgoldnanorodsthatwereconjugatedwith antibodies.Variousaspectsofthesecontrast agents wereresearchedandinvestigated.Sheisnowworking onseveralotherprojectsincludingthephotoacoustic measurement ofthe Gru¨neisenparameterusing an integratingsphere.

WiendeltSteenbergenobtainedaPhDdegreeinfluid dynamicsattheEindhovenUniversityofTechnologyin 1995,afterwhichhejoinedtheUniversityofTwente, Enschede(theNetherlands)asapostdoc.In2000he wasappointedassistantprofessorinbiomedicaloptics andbroadenedhisscopetolow-coherence interferom-etryandphotoacousticandacousto-opticimaging.In 2010,hebecamefullprofessorandgroupleaderofthe newlyformedBiomedicalPhotonicImaginggroupof theUniversityofTwente.