Label-free oxygen-metabolic photoacoustic microscopy in vivo · thesia, Euthanex). After the experiment, the animal naturally recovered and was returned to its cage. For tumor study,

Label-free oxygen-metabolicphotoacoustic microscopy in vivo

Junjie YaoKonstantin I MaslovYu ZhangYounan XiaLihong V Wang

Downloaded from SPIE Digital Library on 18 Jul 2011 to 12825220193 Terms of Use httpspiedlorgterms

Journal of Biomedical Optics 16(7) 076003 (July 2011)

Label-free oxygen-metabolic photoacousticmicroscopy in vivo

Junjie Yao Konstantin I Maslov Yu Zhang Younan Xia and Lihong V WangWashington University in St Louis Department of Biomedical Engineering One Brookings DriveSt Louis Missouri 63130

Abstract Almost all diseases especially cancer and diabetes manifest abnormal oxygen metabolism Accuratelymeasuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies andeven early diagnosis and treatment of disease Current techniques either lack high resolution or rely on exogenouscontrast Here we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution tononinvasively quantify MRO2 in vivo in absolute units mPAM is the unique modality for simultaneously imagingall five anatomical chemical and fluid-dynamic parameters required for such quantification tissue volume vesselcross-section concentration of hemoglobin oxygen saturation of hemoglobin and blood flow speed Hyperther-mia cryotherapy melanoma and glioblastoma were longitudinally imaged in vivo Counterintuitively increasedMRO2 does not necessarily cause hypoxia or increase oxygen extraction In fact early-stage cancer was found tobe hyperoxic despite hypermetabolism Ccopy2011 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI 10111713594786]

Keywords metabolic rate of oxygen photoacoustic imaging hyperthermia cryotherapy tumor hypermetabolism tumor hyperoxia

Paper 11075R received Feb 17 2011 revised manuscript received Apr 12 2011 accepted for publication May 5 2011 publishedonline Jul 1 2011

1 IntroductionThe energy demand of mammals is primarily met by aerobicmetabolism producing 88 of ATP molecules1 Therefore themetabolic rate of oxygen (MRO2) is an important indicator oftissue viability and functionality It is known that nearly allcancers after the early stage are starved for oxygen (hypoxia)due to hypermetabolism andor limited blood supply regard-less of their cellar origins2 In the presence of low oxygenpressure highly malignant cancer cells survive and prolifer-ate via glycolysis (anaerobic respiration the Warburg effect)The presence of a large number of hypoxic regions within atumor usually correlates with a poor prognosis1 This metabolicphenotype has become the basis for tumor imaging by positronemission tomography (PET) using radioactively labeled oxy-gen Many other pathological and physiological functions arealso closely related to alterations of oxygen metabolism ex-amples include Alzheimerrsquos disease3 diabetes4 burns5 ob-structive pulmonary disease6 congestive heart failure7 aging8

sleeping9 and physiologic challenges10 Therefore an accu-rate measurement of MRO2 has the potential to provide apowerful tool for diagnosis and therapy of cancer and otherdiseases as well as for metabolism-related pathophysiologicalstudies

Compared with other oxygenation indexes of tissue ieoxygen saturation (sO2) of hemoglobin and partial oxygenpressure (pO2) MRO2 is superior because it directly reflectsthe rate of oxygen consumption instead of the static oxygenconcentration11 If the region of interest has well-defined feed-

Address all correspondence to Lihong Wang Washington University in StLouis Biomedical Engineering One Brookings Drive Campus Box 1097St Louis Missouri 63130 Tel 314 935 6152 Fax 314 935 7448 E-maillhwangbiomedwustledu

ing and draining vessels we have12

MRO2 = εtimesCHbtimes (sO2intimesAintimesvin minus sO2outtimesAouttimesvout )W

(1)Here subscripts in and out denote feeding and drainingvessels respectively ε is the oxygen binding capacity ofhemoglobin and is usually taken as a constant (136 ml O2gramhemoglobin)13 CHb is the total hemoglobin concentration (ingrams of hemoglobinml blood) sO2 is the average oxygen sat-uration (in ) A is the cross-sectional area (in mm2) v is theaverage blood flow speed (in mms) W is the weight of theregion of interest (in grams) While CHb can be estimated any-where in the trunk vasculature the other parameters must belocally measured

Presently three primary imaging modalities are used to quan-tify MRO212 Among them PET is most widely used in clin-ical practice However the need for injection or inhalationof radioactively labeled exogenous tracers results in a com-plex procedure with exposure to ionizing radiation limiting itsusage14 Functional magnetic resonance imaging (fMRI) hasalso been intensively used for MRO2 study especially in thebrain fMRI is limited to qualitative evaluation of only tem-poral changes in MRO2 and has difficulty in measuring bothCHb and sO215 It also must switch between different imag-ing protocols to measure sO2 and v 16 17 Moreover both PETand fMRI are expensive Diffuse optical tomography (DOT)is also capable of measuring MRO2 and is relatively inexpen-sive but it relies on an approximate theoretical model or othertechniques (eg Doppler ultrasound and laser Doppler) to pro-vide blood flow information18 19 Recently DOT has been com-bined with diffuse correlation spectroscopy (DCS) which iscapable of providing relative blood flow information20 Fur-thermore due to their relatively poor spatial resolutions PET

1083-3668201116(7)07600311$2500 Ccopy 2011 SPIE

Journal of Biomedical Optics July 2011 Vol 16(7)076003-1


Yao et al Label-free oxygen-metabolic photoacoustic microscopy in vivo

fMRI and DOT usually measure MRO2 averaged over a largevolume21

Here we overcome these limitations by developing label-free metabolic photoacoustic microscopy (mPAM) that ultra-sonically measures optical contrast through the photoacoustic(PA) effect For the first time we demonstrated that all fiveanatomic chemical and fluid-dynamic parameters for MRO2

quantification can be obtained in absolute units by mPAM alonein vivo To validate mPAM first we studied the MRO2 responsesto hyperthermia and cryotherapy two common therapeutic tech-niques Furthermore mPAM was used to longitudinally im-age melanoma and glioblastoma demonstrating its capability ofearly cancer detection

2 Materials and Methods21 Ethical Review of ProceduresAll experimental animal procedures were carried out in confor-mity with the laboratory animal protocol approved by the AnimalStudies Committee at Washington University in St Louis

22 Experimental AnimalsThe ears of adult four- to five-week-old nude mice (HsdAthymic Nude-FoxlNU Harlan Co body weight sim20 g) wereused for all in vivo experiments During data acquisition theanimal was held steady with a dentalhard palate fixture and

kept still by using a breathing anesthesia system (E-Z Anes-thesia Euthanex) After the experiment the animal naturallyrecovered and was returned to its cage For tumor study af-ter monitoring the animal was sacrificed by an intraperitonealadministration of pentobarbital at a dosage of 120 mgkg

23 Metabolic Photoacoustic MicroscopyMetabolic photoacoustic microscopy [Fig 1(a)] is based onnewly developed photoacoustic microscopy which has showna robust capability to noninvasively image microvasculature us-ing endogenous contrast with high spatial resolution (lateralsim5 μm axial sim15 μm)22 Briefly speaking a tunable dyelaser (CBR-D Sirah) pumped by a NdYLF laser (INNOSABEdgewave 523 nm) serves as the light source The laser pulseis reshaped by a 25-μm diameter pinhole and focused ontothe surface of the mouse ear by a microscope objective lens(Olympus 4times NA = 01) with a pulse energy of sim100 nJUltrasonic detection is achieved through a spherically focusedultrasonic transducer (V2012-BC Panametrics-NDT) which isconfocally placed with the objective The detected PA signal isthen amplified digitized and saved A volumetric image is gen-erated by recording the time-resolved PA signal (A-line) at eachhorizontal location of the two-dimensional raster scan The mo-tion controller provides the trigger signals for laser firing dataacquisition and motor scanning

(b) (c)(a)

Laser(s)

PinholeVein

1500Objective lens

Laser beam

C (gl)10

sO

SOLCL

CHb (gl) sO2

(d) (e)

8

10Artery

ALUTwave

4

6

8

ed (

mm

s)

Vein

Object

MS

Vein

0

2

4

Sp

ee Veiny

x

z

10-10Speed (mms)

0 200 4000

y (microm)0

Fig 1 Label-free quantification of MRO2 in vivo (a) Schematic of mPAM system Nanosecond laser pulses pass through a pinhole and are focusedon the animalrsquos skin surface by an objective The resultant PA signal is detected by a focused ultrasonic transducer which is confocally placed withthe objective A transverse resolution of 5 μm and an axial resolution of 15 μm are achieved with a penetration depth greater than 700 μm CLcorrection lens AL acoustic lens SQL silicone oil layer UT ultrasonic transducer WT water tank MS motorized scanner (b) mPAM image of thetotal concentration of hemoglobin (CHb) Scale bar 500 μm (c) mPAM image of the sO2 of hemoglobin in the area indicated by the dashed box in(b) Scale bar 125 μm (d) mPAM image of blood flow in the area indicated by the dashed box in (c) Red arrow positive scanning direction bluearrow negative scanning direction Positive velocity upward flow Scale bar 125 μm (e) Profile of blood flow speed across the dashed line in (d)(Color online only)




All of the parameters for MRO2 quantification in Eq (1) canbe simultaneously obtained by mPAM Specifically anatomicparameters W and A are quantified from the structural mPAMimage23 functional parameters CHb and sO2 are measuredby laser excitation at two wavelengths23 24 fluid-dynamic pa-rameter v is estimated on the basis of photoacoustic Dopplerbandwidth broadening of the PA signal induced by circulatingred blood cells25 26 The structural image acquisition time issim25 min for a 4 mm times 4 mm region (sim1 Hz frame rate)using a single wavelength the oxygenation image acquisitiontime is sim20 min for a 1 mm times 1 mm region using two wave-lengths (sim03 Hz frame rate) the flow speed acquisition timeis sim5 min for a 05 mm cross-sectional line using a singlewavelength

24 Hyperthermia Experimental ProtocolIn the hyperthermia study the animalrsquos temperature was reg-ulated by adjusting the water temperature in the heating padplaced underneath its abdomen The water was circulated by awater-bath system (ISOTEMP 9100 Fisher Scientific) A cot-ton layer between the heating pad and the animal skin bufferedand homogenized the temperature change and thus protectedthe animal from burns The room temperature was kept at 23 oCThe animalrsquos skin temperature (SKT) was monitored on the dor-sal pelvis by an attached electronic thermometer (Radio ShackCat No 63-854) Before the experiment the SKT was adjustedto 31 oC which was used as the baseline The experiment wasdivided into three periods The animal was first monitored atbaseline temperature for sim40 min (the resting period) thenheated for sim30 min by increasing the heading pad to 50 oC(the heating period) and last allowed to cool to baseline forsim100 min (the recovery period) The hemodynamic parameterswere simultaneously measured on the principal artery-vein pair(AVP) using mPAM Each measurement took sim6 min and thewhole experiment lasted for sim3 h

25 Tumor Cell CultureB16 mouse melanoma cells were obtained from the TissueCulture and Support Center at the Washington UniversitySchool of Medicine The cells were maintained in Dulbeccorsquosmodified Eagle medium (DMEM Invitrogen Carlsbad Cali-fornia) supplemented with 10 fetal bovine serum (FBS) and1 PS U87 MG human brain glioblastoma cells (HTB-14)were obtained from American Type Cell Culture (ATCC) Thecells were maintained in Eaglersquos Minimum Essential Medium(EMEM Invitrogen) supplemented with 10 heat-inactivatedFBS (ATCC) and 1 penicillin-streptavidin (PS Invitrogen)The cultures were performed at 37 C and 5 CO2 and the cellswere passaged weekly

26 Inoculation of Tumor CellsIn the tumor study 001 ml of suspension containing sim05 mil-lion B16 melanoma cells or U87 human glioblastoma cells wasinoculated into the top skin layer in the left ear of a nudemouse using a 03 ml syringe with a 29-gauge needle Theinjection was usually located near the second order branch ofthe principal AVP above the cartilage The tumor was allowed

to grow and monitored for one to three weeks A control mea-surement was performed before the tumor cell injection (dayzero)

27 Hemodynamics Measured by MetabolicPhotoacoustic Microscopy

The hemodynamic parameters were monitored using mPAMon the principal AVP which included vessel diameter totalhemoglobin concentration oxygen saturation blood flow di-rection and flow speed

1 Vessel diameter The vessel diameter was measuredalong a cross-section perpendicular to the vessel cen-terline A threshold of two times the noise level wasempirically set to separate the background and vesselThe transverse dimension of the vessel cross section wastaken as the vessel diameter D in case the excitationlight could not penetrate the whole vessel in the axialdirection Then the vessel cross-section area was calcu-lated as A = π (D4)2 Five adjacent cross sections weremeasured and the mean value of the principal vein wasused for MRO2 calculation

2 Total hemoglobin concentration Since oxy- and deoxy-hemoglobin have the same absorption coefficient at584 nm the PA signal amplitude is proportional to thetotal hemoglobin concentration regardless of the oxygensaturation level27 On the basis of a previous study28 thetotal hemoglobin concentration was set to be 1460 glat baseline temperature (the hyperthermia study) andon day zero (the tumor and cryotherapy studies) Thesequential data sets were accordingly calibrated The av-eraged PA signal amplitude along the centerline of theprincipal vein was used for calculation

3 Oxygen saturation The sO2 was measured using laserexcitations at 584 and 590 nm respectively24 The rela-tive change in molar extinction coefficient is sim584for oxy- and 175 for deoxy-hemoglobin respec-tively Because the vessels studied here were big enough(gt 50 μm) and the laser pulse energy was low (sim100 nJ)the difference of penetration depth will not affect theresults much The PA signal at each wavelength wasnormalized with the laser pulse energy as monitored bya wavelength-calibrated photodiode The averaged sO2

along the vessel centerline was used for the MRO2 cal-culation The PA image acquired at 584 nm was usedas a mask on the sO2 image to remove the backgroundnoise

4 Blood flow direction The blood flow direction wasmeasured on the basis of photoacoustic Doppler band-width broadening with bidirectional motor scanning at584 nm25 The laser repetition rate was 3 KHz and thescanning step size was 0625 μm We used 64 sequen-tial A-lines to calculate the bandwidth broadening Ifthe blood flow had a positive projection along the posi-tive scanning direction we defined it as a positive flowand vice versa The mPAM image acquired at 584 nmwas used as a mask on the flow image to remove thebackground noise




5 Blood flow speed To better estimate the flow speedM-mode imaging at 584 nm was used to measure thebandwidth broadening across the vessel The laser repe-tition rate was 3 KHz and 3200 A-lines were acquired ateach position The flow speed at each position was calcu-lated on the basis of the Doppler bandwidth broadeningThe average amplitude of the PA signal at each positionwas used as a mask on the speed profile to remove thebackground noise The mean flow speed of the principalvein was used for MRO2 calculation

28 Melanoma Volume Estimation using MetabolicPhotoacoustic Microscopy

After data acquisition the PA signal amplitude acquired ateach optical wavelength was extracted through the Hilberttransformation The tumor region was then separated fromthe surrounding blood vessels according to the mPAM imageacquired at 605 nm where melanin has much stronger absorp-tion than blood A threshold of 20 of the maximum signalamplitude was set to segment the tumor Since it was challengingto penetrate through the whole tumor due to the high absorptionof melanin a 3D envelope of the tumor region was obtained fromthe surface signal instead The volume of the tumor was then cal-

culated by integrating the corresponding voxels enclosed by theenvelope All of the image processing was conducted using theMATLAB Image Processing Toolbox (R2008a MathWorks)

29 Fitting for the Profile of Blood Flow SpeedA theoretical model was used to fit the profile of the blood flowspeed across the vessel29

v(x) = vmaxtimes[

1 minus(

x minus x0

R

)n] (2)

Here x is the transverse location x0 is the vessel center R isthe vessel radius vmax is the flow speed at the vessel center andn is the power index that characterizes the flow profile (eg n= 2 for laminar flow) While x0 and R can be directly measuredfrom the mPAM images of the vessel structure vmax and n arethe unknown parameters to fit for

210 Statistical AnalysisQuantitative data was expressed as mean plusmn sem The statis-tical test is a paired Studentrsquos t-test (two-tailed with unequalvariance) compared with the baseline levels (hyperthermia andcryotherapy studies) or day zero (tumor studies) We considereda p-value less than 005 to be statistically significant

1010

ms

)(a)

A1 V

(b)A1

(c)

08

09

O2

5

pee

d (

mm

A1 V11

V1

Artery

06

07

sO

10

mm

s)

0Sp

A2 V2 A2Vein

1 2 3 405

Vessel order0

5

Sp

eed

(m

V2

A3

V3

5

10

d (

mm

s)

A3V

(d)

25 ArteryVein)

10

s)

AV4

0Sp

eed V3

15

20Vein

te (

lmin

)

5

pee

d (

mm

A4V4

A4V4 05

10

Flow

rat

0 100 200 300 4000S

p

02 1sO2

1 2 3 400

Vessel ordery (microm)

Fig 2 mPAM measurements of sO2 and blood flow on four orders of artery-vein pairs in the mouse ear (a) sO2 mapping from two measurements at584 and 590 nm Four different orders of artery-vein pairs are labeled Scale bar 125 μm (b) Profiles of blood flow speed across vessels of differentorders measured along the dashed lines in (a) on the basis of PA Doppler bandwidth broadening at 584 nm Generally the flow speed decreasesfrom the proximal order to the distal order However it also depends on the vessel diameter as a result of the conservation of total flow Solid circlesexperimental data red curves fit (c) Mean sO2 versus the vessel order quantified along the centerline of each vessel The sO2 values in both thearteries and veins slightly change with the vessel order especially at distal branches This is probably due to oxygen diffusion between the arteriesand veins and the inhomogeneous MRO2 (d) Blood flow rate (in μlmin) versus the vessel order quantified from the flow speed profiles and vesseldiameters Blood flow in both arteries and veins decreases from the proximal order to the distal order and is conserved between the artery and veinof the same order The blood fed by the artery is mostly drained by the corresponding vein of the same order V vein A artery (Color online only)




3 Results31 Metabolic Rate of Oxygen Quantification

under NormothermiaThe nude mouse ear is a good model for validating mPAMbecause of its similarity to human skin and lack of motionartifacts30ndash32 Each artery-vein pair (AVP) feeds a well-definedregion while one pair at the base of the ear feeds the entireear (Fig 2)33 34 Consequently the MRO2 based on each AVPapproximates the MRO2 of its supplied region

As an example we measured the MRO2 of a mouse earunder normothermia The animalrsquos temperature was regulatedwith a heating pad placed under its abdomen (skin temperature31 oC) and a volumetric image was acquired using mPAM at584 nm by scanning a 10 mm times 8 mm area containing the prin-cipal AVP [Fig 1(b)] Because 584 nm is an isosbestic wave-length for hemoglobin this image maps the concentration oftotal hemoglobin regardless of the oxygen saturation level Inaddition it measures the diameters of the principal AVP (arterysim65 μm vein sim116 μm) Two PAM images acquired at 584and 590 nm were then used to calculate sO2 [Fig 1(c)]23 Thevessels with high sO2 values (gt90) are classified as arter-ies whereas the vessels with low sO2 values (60 to 80) areveins The blood flow velocity was measured at 584 nm usingbi-directional scanning with a laser repetition rate of 3 KHz anda motor step size of 0625 μm [Fig 1(d)] The profile of flowspeed across the principal AVP is shown in Fig 1(e) The artery

and the vein have a mean flow speed of 55 and 18 mms respec-tively and the speed profiles are both approximately parabolic29

The weight of the mouse ear was computed by its volume in the3D PA image where the average specific weight was assumedto be 10 gml35 From these measurements the MRO2 of themouse ear was estimated to be 023 ml100 gmin which agreeswith the data previously measured in humans2

32 Change in Metabolic Rate of Oxygen Inducedby Systemic Hyperthermia

Hyperthermia has been clinically used for cancer treatment36

To measure MRO2 during hyperthermia the mousersquos skin tem-perature was elevated to 42 oC [Fig 3(a)] Hemodynamics weremonitored on the principal of AVP The vessel diameter startedincreasing at the beginning of the heating period [Fig 3(b)]and reached a maximum by the end of the heating period Thetotal hemoglobin concentration of the principal AVP kept in-creasing after the heating started and plateaued when the tem-perature returned to the baseline [Fig 3(c)] This cumulativeeffect was due to a decrease in blood plasma volume resultingfrom water loss during hyperthermia37 From the change in sO2

[Fig 3(d)] we found that the oxygen extraction fraction (OEFdefined as (sO2in minus sO2out )sO2in and represents the fraction ofO2 molecules that cross the capillary wall) decreased by 12over the heating period and eventually recovered to 99 of theresting level [Fig 3(f)] The measurements of flow speed in the

Skin temperature60

Vessel diameter15

CHb20

(b) )c()a(

V i V i

20

40

e ch

ange

()

5

10

e ch

ange

()

10

15

e ch

ange

()Vein

Artery

VeinArtery

Heating

0 40 80 120 160-20

0

Rel

ativ

e

0 40 80 120 160-5

0

Rel

ativ

e

0 40 80 120 160-5

0

5

Rel

ativ

e

0 40 80 120 160Time (min)

0 40 80 120 160Time (min)

Blood flow speed60

OEF and MRO260

0 40 80 120 160Time (min)

sO28

)f()e()d(

V iV i

40

60

chan

ge (

)

20

40

60

chan

ge (

)

2

4

6

8

chan

ge (

)

OEFMRO2

VeinArtery

VeinArtery

0

20

Rel

ativ

e

-40

-20

0

Rel

ativ

e c

-4

-2

0

2

Rel

ativ

e c

0 40 80 120 160Time (min)

0 40 80 120 16040

Time (min)0 40 80 120 160

4

Time (min)

Fig 3 mPAM measurement of hemodynamic responses to a hyperthermic challenge (red bars) Relative changes of (a) the skin temperature (b)vessel diameter (c) total hemoglobin concentration (d) sO2 (e) blood flow speed and (f) OEF and MRO2 Statistics paired Studentrsquos t-test plt 005 p lt 001 n = 3 Data are presented as means plusmn sem The baseline values are (a) temperature 310 plusmn 04 oC (b) vessel diameters925 plusmn 105 μm (vein) and 475 plusmn 85 μm (vein) (c) CHb 1463 plusmn 181 gl (vein) and 1485 plusmn 140 gl (artery) (d) sO2 070 plusmn 008 (vein) and094 plusmn 004 (artery) (e) flow speeds 13 plusmn 04 mms (vein) and 51 plusmn 08 mms (artery) and (f) 026 plusmn 013 (OEF) and 030 plusmn 009 ml100 gmin(MRO2) (Color online only)




BaselineVasodilatation

(day 0)Inflammation

(day 12)Necrosis

(day 3)Recovery(day 30)(a)

150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB

Fig 4 mPAM monitoring of hemodynamic responses after cryotherapy (a) mPAM vasculature images acquired in different phases before and afterthe application of liquid nitrogen The treated area is indicated by cyan dashed circles Scale bar 500 μm (b) mPAM images of sO2 in the artery-veinpair [cyan dashed box in (a)] that supports the treated area Scale bar 125 μm (c) mPAM quantification of blood flow rate OEF and MRO2 within30 days after the treatment A neighboring area close to the treated region was monitored as a control Statistics paired Studentrsquos t-test p lt 005 p lt 001 p lt 0001 n = 4 Data are presented as means plusmn sem Baseline flow rates 072 plusmn 012 μlmin (treated) and 044 plusmn 015 μlmin(control) baseline OEF 035 plusmn 004 (treated) and 031 plusmn 005 (control) baseline MRO2 030 plusmn 006 ml100 gmin (treated) and 025 plusmn 007ml100 gmin (control) BL baseline (Color online only)

Day 0 Day 3 Day 7(a) (c)30e

MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)

Fig 5 mPAM detection of early-stage melanoma by measuring MRO2 (a) White-light photographs of a representative mouse ear before (day 0)three and seven days after the xenotransplantation of B16 melanoma tumor cells Scale bar 1 mm (b) mPAM images of the tumor region [dashedboxes in (a)] at 584 nm z is coded by colors blue (superficial) to red (deep) Scale bar 125 μm (c) mPAM quantification of blood flow rateOEF and MRO2 before (day zero) three and seven days after the tumor xenotransplantation normalized by the values of day zero (flow rate 177plusmn 050 μlmin OEF 031 plusmn 004 MRO2 038 plusmn 003 ml100 gmin) Statistics paired Studentrsquos t-test p lt 001 p lt 0001 n = 5 Dataare presented as means plusmn sem MT melanoma tumor VD vasodilatation (Color online only)




60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)

Fig 6 mPAM characterization of melanoma progression mPAM measurements in absolute units of (a) ear weight (b) vessel diameter (c) totalhemoglobin concentration (d) sO2 of hemoglobin (e) volumetric blood flow rate (f) OEF (g) rate of O2 consumption in μlmin and (h) MRO2in ml100 gmin up to 21 days after tumor inoculation Statistics paired Studentrsquos t-test p lt 005 p lt 001 p lt 0001 n = 3 Data arepresented as means plusmn sem

arteries during the heating were saturated because of the limitedmaximum measurable speed of the system [Fig 3(e)] From themeasurements on the principal veins we found the volumetricflow rate of blood entering the ear increased by 45 Increasedcardiac output and redistribution of blood to the skin are twomajor reasons for vessel dilation and faster blood flow whichhelp accelerate heat exchange with the environment36 38 Notethat the vessel diameter sO2 and blood flow speed reach thepeaks approximately simultaneously

The MRO2 of the mouse ear as computed from the hemo-dynamic changes increased by 28 over the heating period[Fig 3(f)] which indicated elevated oxygen metabolism dur-ing hyperthermia in response to an increased rate of enzymaticreactions39 This finding can potentially elucidate another pos-sible mechanism for cell death induced by hyperthermia Whennormal cells encounter such an increased metabolism increasedblood flow provides more nutrients By contrast cancer cellscould be damaged owing to inadequate blood circulation There-fore hyperthermia may kill cancer cells by both protein denat-uration and cell starvation due to heating

33 Change in Metabolic Rate of Oxygen Inducedby Local Cryotherapy

Cryotherapy has been found effective for treating cancer andother diseases by forming ice crystals inside cells40 41 Here weapplied liquid nitrogen to the mouse ear surface for 10 s via a1-mm diameter cryo-probe and monitored the hemodynamics ofthe treated area [Figs 4(a) and 4(b)] An untreated neighboringarea of the same ear was also monitored as a control Right afterthe treatment a global reflective vasodilatation was observed onboth the treated and control areas which was accompanied by anincrease in blood flow and a decrease in OEF While the MRO2

Day 0 Day 3(a) (b)Photo mPAM

MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)

Fig 7 mPAM differentiation of blood vessels and melanoma (a)White-light photographs of a representative mouse ear before andthree days after the xenotransplantation of B16 melanoma cells Scalebar 1 mm (b) Composite mPAM images of blood vessels (in red)and melanoma (in brown) The two are differentiated by using dual-wavelength excitation at 584 and 605 nm CHb total hemoglobinconcentration CM melanin concentration Scale bar 250 μm MTmelanoma tumor (Color online only)




(a) (b)

MT

050

Fig 8 Histological validation of melanoma detection (a) mPAM im-age of a mouse ear bearing a B16 melanoma tumor acquired on dayseven z is coded by colors blue (superficial) to red (deep) Scale bar300 μm (b) Images of HampE stained tissue slices cut approximatelyacross the tumor area (top) and nontumor area (bottom) Scale bar150 μm MT melanoma tumor (Color online only)

of the control area remained statistically unchanged the MRO2

of the treated area decreased by 56 due to the induced celldeath Therefore mPAM can be used to evaluate the efficacy ofcryotherapy Within one month after the treatment while all ofthe parameters of the control area monotonically recovered to thebaseline the physiological progress of the treated area occurredin phases [Fig 4(c)] Within three days following the reflectivevasodilatation blood flow and OEF trended toward the baselinebut MRO2 remained at a low level due to cell necrosis Startingfrom day five inflammation was clearly observed which was

triggered by the immune system and was helpful for both deadcell clearance and new cell growth Although the OEF continuedto decrease due to the increased flow speed11 the MRO2 ofthe treated area eventually returned to the baseline reflectingimproved tissue viability One month later the inflammationnearly ended and all of the parameters had recovered almost tothe baseline

This study shows that each physiological phase aftercryotherapy imparts its signature on the local MRO2 Thecommon belief is that inflammation triggered by the immuneresponse further helps kill tumor cells42 However our resultsshow that the increased blood flow rate during inflammationmay assist the survival of residual tumor cells by providingmore nutrients and thus recovering the MRO2 level

34 Early Cancer Detection by MeasuringTumor-induced Change in Metabolic Rateof Oxygen

The third demonstration of mPAM is early cancer detection bymeasuring MRO2 The hemodynamics of the mouse ear werelongitudinally monitored after the injection of B16 melanomacells [Fig 5(a)] On day 7 vessel dilation appeared aroundthe tumor site as shown in Figs 5(b) and 6(b) The volumetricblood flow rate increased by 15 fold [Figs 5(c) and 6(e)] Thesechanges are important to ensure the supply of oxygen and nutri-ents to the rapidly growing tumor and to provide routes for tumorcell metastasis43 The overall OEF of the tumor region decreasedby 43 [Figs 5(c) and 6(f)] due to the increased blood flow11

The vasculature and melanoma were differentiated according to

(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2

Fig 9 mPAM detection of early-stage glioblastoma by measuring MRO2 (a) White-light photographs of a representative mouse ear before (dayzero) and seven days after the xenotransplantation of U87 glioblastoma tumor cells Scale bar 2 mm (b) mPAM images of microvasculature in thetumor region [dashed boxes in (a)] at 584 nm z is coded by colors blue (superficial) to red (deep) Scale bar 250 μm (c) mPAM images of sO2in the artery-vein pair [double arrows in (b)] that supports the tumor region acquired on day zero and day seven Scale bar 100 μm (d) mPAMquantification of volumetric blood flow rate OEF and MRO2 seven days after the tumor xenotransplantation normalized by the values of day zero(flow rate 103 plusmn 041 μlmin OEF 027 plusmn 003 MRO2 031 plusmn 009 ml100 gmin) (e) Comparison of the averaged sO2 values in the intra- andextra-tumoral vasculatures Statistics paired Studentrsquos t-test p lt 005 p lt 001 n = 5 Data are presented as means plusmn sem GT glioblastomatumor (Color online only)




(a) 05

m)

GT

z(m

m

(b)0

GT

Fig 10 Histological validation of glioblastoma detection (a) mPAM image of a mouse ear bearing a U87 glioblastoma tumor acquired on dayseven z is coded by colors blue (superficial) to red (deep) Scale bar 250 μm (b) Images of HampE stained tissue slices cut approximately across thetumor (left) and nontumor areas (right) Scale bar 150 μm GT glioblastoma tumor (Color online only)

their different absorption spectra using dual-wavelength excita-tion at 584 and 605 nm and thus the tumor volume could beestimated (Fig 7) The hypermetabolism of melanoma was re-flected by a 36 increase in MRO2 [Fig 5(c)] which proves theearly cancer detection capability of mPAM The presence of themelanoma was confirmed by histology (Fig 8) However themelanoma was hyperoxic instead of hypoxic in the early stage[Fig 6(d)] On day 14 MRO2 dropped to the baseline level andcontinued to decrease [Fig 6(h)] even though the total oxygenconsumption rate steadily increased [Fig 6(g)] There are twopossible reasons for the final decline in MRO2 On one hand atumor changes to anaerobic respiration instead of aerobic respi-ration when it grows too quickly to get sufficient oxygen2 on theother hand when the tumor grows too large the tumor core diesdue to a decrease in available nutrients (necrosis) The necrotictumor core does not consume oxygen but increases tumor weight[Fig 6(a)] which decreases the MRO244

Besides melanoma we also studied U87 human glioblastoma[Fig 9(a)] which is more transparent thus its intratumoral vas-culature can be better visualized On day seven angiogenesiswas observed within the tumor region [Fig 9(b)] and the sO2

of the draining vein was found to be increased indirectly indi-cating early-stage tumor hyperoxia [Fig 9(c)] The presence ofthe glioblastoma was confirmed by histology (Fig 10) Whilethe increase in blood supply for the glioblastoma was compara-ble with that for the melanoma the OEF showed a decrease by24 instead of 43 [Fig 9(d)] We observed a 100 increasein MRO2 instead of 36 for the melanoma which indicateda stronger hypermetabolism at the early stage of glioblastomaHowever characteristic of early-stage cancer45 such hyperme-tabolism did not lead to tumor hypoxia In fact the sO2 in theintratumoral vasculature was even higher than that of the sur-rounding normal tissue directly indicating early-stage tumorhyperoxia [Fig 9(e)] The increase in sO2 actually caused thedecrease in OEF in the tumor This observation suggests that ahypoxia-based diagnosis may not apply to early-stage cancer46

4 Conclusions and DiscussionThe observations presented here demonstrate the power ofmPAM as the only noninvasive label-free imaging modality thatcan measure all of the parameters required for the quantificationof MRO2 in absolute units Whereas MRO2 is the ultimate mea-sure of oxygen metabolism OEF and sO2 can be misleadingpartial measures Unlike as is commonly believed a decreasein OEF or an increase in sO2 does not necessarily indicate adecrease in MRO2 Strikingly we found early-stage cancer tobe hyperoxic instead of hypoxic despite the hypermetabolism

mPAM can noninvasively measure anatomical functionaland fluid-dynamic information at the resolution of small ves-sels making it possible for MRO2 quantification in microenvi-ronments MRO2-based early cancer detection and evaluationof its treatment are highly desirable mPAM also has variousother prospective applications related to MRO2 First its highspatial resolution is essential for micro-hemodynamic studiessuch as monitoring of local hemorrhage caused by mini-strokesSecond its high sensitivity is critical for studies concerningsmall metabolic changes such as the monitoring of neuro-vascular coupling in response to physiological challenges Thirdits potentially real-time imaging through fast optical scanningor ultrasonic-array detection is important for studies involvingshort transition times between physiological states eg mon-itoring of epileptic seizures Finally its high spatial scalabil-ity enables us to correlate microscopic and macroscopic studies(eg monitoring of local neuron firing and overall brain activity)based on the same contrast Overall mPAM has strong poten-tial for the study of metabolism in cancer and other metabolicdiseases

AcknowledgmentsThe authors thank Christopher Favazza Kim Chulhong SongHu Lidai Wang Dakang Yao and Arie Krumholz for helpfuldiscussions Li Li for experimental assistance and Professor




James Ballard for manuscript editing This research was sup-ported by the National Institutes of Health Grants Nos R01EB000712 R01 EB008085 R01 CA134539 U54 CA136398R01 EB010049 and 5P60 DK02057933

References1 T N Seyfried and L M Shelton ldquoCancer as a metabolic diseaserdquo

Nutr Metab 7(7) 1ndash22 (2010)2 P Vaupel F Kallinowski and P Okunieff ldquoBlood-flow oxygen and

nutrient supply and metabolic microenvironment of human-tumors ndash areviewrdquo Cancer Res 49(23) 6449ndash6465 (1989)

3 X Zhang and W D Le ldquoPathological role of hypoxia in Alzheimerrsquosdiseaserdquo Exp Neurol 223(2) 299ndash303 (2010)

4 K Cheng K Ho R Stokes C Scott S M Lau W J HawthorneP J OrsquoConnell T Loudovaris T W Kay R N Kulkarni T OkadaX H L Wang S H Yim Y Shah S T Grey A V Biankin J GKench D R Laybutt F J Gonzalez C R Kahn and J E GuntonldquoHypoxia-inducible factor-1 alpha regulates beta cell function in mouseand human isletsrdquo J Clin Invest 120(6) 2171ndash2183 (2010)

5 T Tadros D L Traber and D N Herndon ldquoHepatic blood flowand oxygen consumption after burn and sepsisrdquo J Trauma Inj InfectCrit Care 49(1) 101ndash108 (2000)

6 M Donahoe R M Rogers D O Wilson and B E PennockldquoOxygen-consumption of the respiratory muscles in normal and in mal-nourished patients with chronic obstructive pulmonary-diseaserdquo AmRev Respir Dis 140(2) 385ndash391 (1989)

7 R M Bersin C Wolfe M Kwasman D Lau C Klinski KTanaka P Khorrami G N Henderson T Demarco and K Chat-terjee ldquoImproved hemodynamic function and mechanical efficiencyin congestive-heart-failure with sodium dichloroacetaterdquo J Am CollCardiol 23(7) 1617ndash1624 (1994)

8 G Marchal P Rioux M C Petittaboue G Sette J M Travere CLepoec P Courtheoux J M Derlon and J C Baron ldquoRegional cere-bral oxygen-consumption blood-flow and blood-volume in healthy-human agingrdquo Arch Neurol 49(10) 1013ndash1020 (1992)

9 P Maquet ldquoSleep function(s) and cerebral metabolismrdquo Behav BrainRes 69(1ndash2) 75ndash83 (1995)

10 A de la Zerda Z A Liu S Bodapati R Teed S Vaithilingam BT Khuri-Yakub X Y Chen H J Dai and S S Gambhir ldquoUltrahighsensitivity carbon nanotube agents for photoacoustic molecular imagingin living micerdquo Nano Lett 10(6) 2168ndash2172 (2010)

11 R B Buxton and L R Frank ldquoA model for the coupling betweencerebral blood flow and oxygen metabolism during neural stimulationrdquoJ Cereb Blood Flow Metab 17(1) 64ndash72 (1997)

12 L V Wang ldquoProspects of photoacoustic tomographyrdquo Med Phys35(12) 5758ndash5767 (2008)

13 A C Guyton and J E Hall Textbook of Medical Physiology (ElsevierSaunders Elsevier Science distributor Edinburgh Oxford (2006)

14 R S J Frackowiak G L Lenzi T Jones and J D HeatherldquoQuantitative measurement of regional cerebral blood-flow and oxygen-metabolism in man using O-15 and positron emission tomography ndashtheory procedure and normal valuesrdquo J Comput Assist Tomogr 4(6)727ndash736 (1980)

15 R D Hoge J Atkinson B Gill G R Crelier S Marrett and GB Pike ldquoInvestigation of BOLD signal dependence on cerebral bloodflow and oxygen consumption the deoxyhemoglobin dilution modelrdquoMagn Reson Med 42(5) 849ndash863 (1999)

16 F Xu Y L Ge and H Z Lu ldquoNoninvasive quantification of whole-brain cerebral metabolic rate of oxygen (CMRO2) by MRIrdquo MagnReson Med 62(1) 141ndash148 (2009)

17 V Jain M C Langham and F W Wehrli ldquoMRI estimation of globalbrain oxygen consumption raterdquo J Cereb Blood Flow Metab 30(9)1598ndash1607 (2010)

18 D W Brown J Hadway and T Y Lee ldquoNear-infrared spectroscopymeasurement of oxygen extraction fraction and cerebral metabolicrate of oxygen in newborn pigletsrdquo Pediatr Res 54(6) 861ndash867(2003)

19 D A Boas G Strangman J P Culver R D Hoge GJasdzewski R A Poldrack B R Rosen and J B Mandev-ille ldquoCan the cerebral metabolic rate of oxygen be estimated with

near-infrared spectroscopyrdquo Phys Med Biol 48(15) 2405ndash2418(2003)

20 C Zhou R Choe N Shah T Durduran G Yu A Durkin DHsiang R Mehta J Butler A Cerussi B J Tromberg and AG Yodh ldquoDiffuse optical monitoring of blood flow and oxygenation inhuman breast cancer during early stages of neoadjuvant chemotherapyrdquoJ Biomed Opt 12(5) 051903 (2007)

21 S Hu and L V Wang ldquoPhotoacoustic imaging and characterizationof the microvasculaturerdquo J Biomed Opt 15(1) 011101 (2010)

22 K Maslov H F Zhang S Hu and L V Wang ldquoOptical-resolutionphotoacoustic microscopy for in vivo imaging of single capillariesrdquoOpt Lett 33(9) 929ndash931 (2008)

23 S Hu K Maslov V Tsytsarev and L V Wang ldquoFunctional transcra-nial brain imaging by optical-resolution photoacoustic microscopyrdquo JBiomed Opt 14(4) 040503 (2009)

24 H F Zhang K Maslov G Stoica and L H V Wang ldquoFunctionalphotoacoustic microscopy for high-resolution and noninvasive in vivoimagingrdquo Nat Biotechnol 24(7) 848ndash851 (2006)

25 J Yao K I Maslov Y Shi L A Taber and L V Wang ldquoInvivo photoacoustic imaging of transverse blood flow by using Dopplerbroadening of bandwidthrdquo Opt Lett 35(9) 1419ndash1421 (2010)

26 J J Yao and L H V Wang ldquoTransverse flow imaging based onphotoacoustic Doppler bandwidth broadeningrdquo J Biomed Opt 15(2)021304 (2010)

27 M Sivaramakrishnan K Maslov H F Zhang G Stoica and LV Wang ldquoLimitations of quantitative photoacoustic measurements ofblood oxygenation in small vesselsrdquo Phys Med Biol 52(5) 1349ndash1361(2007)

28 E S Russell E F Neufeld and C T Higgins ldquoComparison of normalblood picture of young adults from 18 inbred strains of micerdquo ProcSoc Exp Biol Med 78(3) 761ndash766 (1951)

29 R S C Cobbold Foundations of Biomedical Ultrasound (OxfordUniversity New York 2007)

30 E J Rebar Y Huang R Hickey A K Nath D Meoli S Nath BL Chen L Xu Y X Liang A C Jamieson L Zhang S K SprattC C Case A Wolffe and F J Giordano ldquoInduction of angiogenesisin a mouse model using engineered transcription factorsrdquo Nat Med8(12) 1427ndash1432 (2002)

31 R K Jain K Schlenger M Hockel and F Yuan ldquoQuantitativeangiogenesis assays progress and problemsrdquo Nat Med 3(11) 1203ndash1208 (1997)

32 T Tammela G Zarkada E Wallgard A Murtomaki S SuchtingM Wirzenius M Waltari M Hellstrom T Schomber R PeltonenC Freitas A Duarte H Isoniemi P Laakkonen G Christofori SYla-Herttuala M Shibuya B Pytowski A Eichmann C Betsholtzand K Alitalo ldquoBlocking VEGFR-3 suppresses angiogenic sprout-ing and vascular network formationrdquo Nature 454(7204) 656ndashU668(2008)

33 E Eriksson J V Boykin and R N Pittman ldquoMethod for in vivomicroscopy of the cutaneous microcirculation of the hairless mouseearrdquo Microvasc Res 19(3) 374ndash379 (1980)

34 J H Barker F Hammersen I Bondar E Uhl T J Galla MD Menger and K Messmer ldquoThe hairless mouse ear for in vivostudies of skin microcirculationrdquo Plast Reconstr Surg 83(6) 948ndash959(1989)

35 M Rins I Diez A C Calpena and R Obach ldquoSkin density inthe hairless rat Evidence of regional differencesrdquo Eur J Drug MetabPharmacokinet 3 456ndash457 (1991)

36 T E Dudar and R K Jain ldquoDifferential response of normal andtumor microcirculation to hyperthermiardquo Cancer Res 44(2) 605ndash612(1984)

37 S G Rhind G A Gannon P N Shek I K M Brenner YSevers J Zamecnik A Buguet V M Natale R J Shephardand M W Radomski ldquoContribution of exertional hyperthermia tosympathoadrenal-mediated lymphocyte subset redistributionrdquo J ApplPhysiol 87(3) 1178ndash1185 (1999)

38 N Charkoudian ldquoSkin blood flow in adult human thermoregulationhow it works when it does not and whyrdquo Mayo Clin Proc 78(5)603ndash612 (2003)

39 R T Pettigre J M Galt C M Ludgate D B Horn and A N SmithldquoCirculatory and biochemical effects of whole-body hyperthermiardquoBrit J Surg 61(9) 727ndash730 (1974)




40 P Perrotte J Tran E J McGuire and L L Pisters ldquoEffect of priortherapy on survival after salvage cryotherapy for recurrent prostatecancerrdquo Prostate Cancer Prostatic Dis 2(S3) S25ndashS25 (1999)

41 P J A Holt ldquoCryotherapy for skin-cancer ndash results over a 5-year pe-riod using liquid-nitrogen spray cryosurgeryrdquo Br J Dermatol 119(2)231ndash240 (1988)

42 J J A Joosten G N P van Muijen T Wobbes and T J M Ruers ldquoInvivo destruction of tumor tissue by cryoablation can induce inhibitionof secondary tumor growth an experimental studyrdquo Cryobiology 42(1)49ndash58 (2001)

43 G H Mahabeleshwar and T V Byzova ldquoAngiogenesis in melanomardquoSemin Oncol 34(6) 555ndash565 (2007)

44 R M Sutherland ldquoCell and environment interactions in tumor microre-gions ndash the multicell spheroid modelrdquo Science 240(4849) 177ndash184(1988)

45 A L Harris ldquoHypoxia ndash A key regulatory factor in tumour growthrdquoNat Rev Cancer 2(1) 38ndash47 (2002)

46 J Pouyssegur F Dayan and N M Mazure ldquoHypoxia sig-nalling in cancer and approaches to enforce tumour regressionrdquo Nature441(7092) 437ndash443 (2006)



Journal of Biomedical Optics 16(7) 076003 (July 2011)

Label-free oxygen-metabolic photoacousticmicroscopy in vivo

Junjie Yao Konstantin I Maslov Yu Zhang Younan Xia and Lihong V WangWashington University in St Louis Department of Biomedical Engineering One Brookings DriveSt Louis Missouri 63130

Abstract Almost all diseases especially cancer and diabetes manifest abnormal oxygen metabolism Accuratelymeasuring the metabolic rate of oxygen (MRO2) can be helpful for fundamental pathophysiological studies andeven early diagnosis and treatment of disease Current techniques either lack high resolution or rely on exogenouscontrast Here we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution tononinvasively quantify MRO2 in vivo in absolute units mPAM is the unique modality for simultaneously imagingall five anatomical chemical and fluid-dynamic parameters required for such quantification tissue volume vesselcross-section concentration of hemoglobin oxygen saturation of hemoglobin and blood flow speed Hyperther-mia cryotherapy melanoma and glioblastoma were longitudinally imaged in vivo Counterintuitively increasedMRO2 does not necessarily cause hypoxia or increase oxygen extraction In fact early-stage cancer was found tobe hyperoxic despite hypermetabolism Ccopy2011 Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI 10111713594786]

Keywords metabolic rate of oxygen photoacoustic imaging hyperthermia cryotherapy tumor hypermetabolism tumor hyperoxia

Paper 11075R received Feb 17 2011 revised manuscript received Apr 12 2011 accepted for publication May 5 2011 publishedonline Jul 1 2011

1 IntroductionThe energy demand of mammals is primarily met by aerobicmetabolism producing 88 of ATP molecules1 Therefore themetabolic rate of oxygen (MRO2) is an important indicator oftissue viability and functionality It is known that nearly allcancers after the early stage are starved for oxygen (hypoxia)due to hypermetabolism andor limited blood supply regard-less of their cellar origins2 In the presence of low oxygenpressure highly malignant cancer cells survive and prolifer-ate via glycolysis (anaerobic respiration the Warburg effect)The presence of a large number of hypoxic regions within atumor usually correlates with a poor prognosis1 This metabolicphenotype has become the basis for tumor imaging by positronemission tomography (PET) using radioactively labeled oxy-gen Many other pathological and physiological functions arealso closely related to alterations of oxygen metabolism ex-amples include Alzheimerrsquos disease3 diabetes4 burns5 ob-structive pulmonary disease6 congestive heart failure7 aging8

sleeping9 and physiologic challenges10 Therefore an accu-rate measurement of MRO2 has the potential to provide apowerful tool for diagnosis and therapy of cancer and otherdiseases as well as for metabolism-related pathophysiologicalstudies

Compared with other oxygenation indexes of tissue ieoxygen saturation (sO2) of hemoglobin and partial oxygenpressure (pO2) MRO2 is superior because it directly reflectsthe rate of oxygen consumption instead of the static oxygenconcentration11 If the region of interest has well-defined feed-

Address all correspondence to Lihong Wang Washington University in StLouis Biomedical Engineering One Brookings Drive Campus Box 1097St Louis Missouri 63130 Tel 314 935 6152 Fax 314 935 7448 E-maillhwangbiomedwustledu

ing and draining vessels we have12

MRO2 = εtimesCHbtimes (sO2intimesAintimesvin minus sO2outtimesAouttimesvout )W

(1)Here subscripts in and out denote feeding and drainingvessels respectively ε is the oxygen binding capacity ofhemoglobin and is usually taken as a constant (136 ml O2gramhemoglobin)13 CHb is the total hemoglobin concentration (ingrams of hemoglobinml blood) sO2 is the average oxygen sat-uration (in ) A is the cross-sectional area (in mm2) v is theaverage blood flow speed (in mms) W is the weight of theregion of interest (in grams) While CHb can be estimated any-where in the trunk vasculature the other parameters must belocally measured

Presently three primary imaging modalities are used to quan-tify MRO212 Among them PET is most widely used in clin-ical practice However the need for injection or inhalationof radioactively labeled exogenous tracers results in a com-plex procedure with exposure to ionizing radiation limiting itsusage14 Functional magnetic resonance imaging (fMRI) hasalso been intensively used for MRO2 study especially in thebrain fMRI is limited to qualitative evaluation of only tem-poral changes in MRO2 and has difficulty in measuring bothCHb and sO215 It also must switch between different imag-ing protocols to measure sO2 and v 16 17 Moreover both PETand fMRI are expensive Diffuse optical tomography (DOT)is also capable of measuring MRO2 and is relatively inexpen-sive but it relies on an approximate theoretical model or othertechniques (eg Doppler ultrasound and laser Doppler) to pro-vide blood flow information18 19 Recently DOT has been com-bined with diffuse correlation spectroscopy (DCS) which iscapable of providing relative blood flow information20 Fur-thermore due to their relatively poor spatial resolutions PET

1083-3668201116(7)07600311$2500 Ccopy 2011 SPIE











(b) (c)(a)

Laser(s)

PinholeVein

1500Objective lens

Laser beam

C (gl)10

sO

SOLCL

CHb (gl) sO2

(d) (e)

8

10Artery

ALUTwave

4

6

8

ed (

mm

s)

Vein

Object

MS

Vein

0

2

4

Sp

ee Veiny

x

z

10-10Speed (mms)

0 200 4000

y (microm)0

























v(x) = vmaxtimes[

1 minus(

x minus x0

R

)n] (2)



1010

ms

)(a)

A1 V

(b)A1

(c)

08

09

O2

5

pee

d (

mm

A1 V11

V1

Artery

06

07

sO

10

mm

s)

0Sp

A2 V2 A2Vein

1 2 3 405

Vessel order0

5

Sp

eed

(m

V2

A3

V3

5

10

d (

mm

s)

A3V

(d)

25 ArteryVein)

10

s)

AV4

0Sp

eed V3

15

20Vein

te (

lmin

)

5

pee

d (

mm

A4V4

A4V4 05

10

Flow

rat

0 100 200 300 4000S

p

02 1sO2

1 2 3 400















Skin temperature60

Vessel diameter15

CHb20

(b) )c()a(

V i V i

20

40

e ch

ange

()

5

10

e ch

ange

()

10

15

e ch

ange

()Vein

Artery

VeinArtery

Heating

0 40 80 120 160-20

0

Rel

ativ

e

0 40 80 120 160-5

0

Rel

ativ

e

0 40 80 120 160-5

0

5

Rel

ativ

e

0 40 80 120 160Time (min)

0 40 80 120 160Time (min)

Blood flow speed60

OEF and MRO260

0 40 80 120 160Time (min)

sO28

)f()e()d(

V iV i

40

60

chan

ge (

)

20

40

60

chan

ge (

)

2

4

6

8

chan

ge (

)

OEFMRO2

VeinArtery

VeinArtery

0

20

Rel

ativ

e

-40

-20

0

Rel

ativ

e c

-4

-2

0

2

Rel

ativ

e c

0 40 80 120 160Time (min)

0 40 80 120 16040

Time (min)0 40 80 120 160

4

Time (min)






(day 0)Inflammation

(day 12)Necrosis


150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB



MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)





60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT









































































(b) (c)(a)

Laser(s)

PinholeVein

1500Objective lens

Laser beam

C (gl)10

sO

SOLCL

CHb (gl) sO2

(d) (e)

8

10Artery

ALUTwave

4

6

8

ed (

mm

s)

Vein

Object

MS

Vein

0

2

4

Sp

ee Veiny

x

z

10-10Speed (mms)

0 200 4000

y (microm)0

























v(x) = vmaxtimes[

1 minus(

x minus x0

R

)n] (2)



1010

ms

)(a)

A1 V

(b)A1

(c)

08

09

O2

5

pee

d (

mm

A1 V11

V1

Artery

06

07

sO

10

mm

s)

0Sp

A2 V2 A2Vein

1 2 3 405

Vessel order0

5

Sp

eed

(m

V2

A3

V3

5

10

d (

mm

s)

A3V

(d)

25 ArteryVein)

10

s)

AV4

0Sp

eed V3

15

20Vein

te (

lmin

)

5

pee

d (

mm

A4V4

A4V4 05

10

Flow

rat

0 100 200 300 4000S

p

02 1sO2

1 2 3 400















Skin temperature60

Vessel diameter15

CHb20

(b) )c()a(

V i V i

20

40

e ch

ange

()

5

10

e ch

ange

()

10

15

e ch

ange

()Vein

Artery

VeinArtery

Heating

0 40 80 120 160-20

0

Rel

ativ

e

0 40 80 120 160-5

0

Rel

ativ

e

0 40 80 120 160-5

0

5

Rel

ativ

e

0 40 80 120 160Time (min)

0 40 80 120 160Time (min)

Blood flow speed60

OEF and MRO260

0 40 80 120 160Time (min)

sO28

)f()e()d(

V iV i

40

60

chan

ge (

)

20

40

60

chan

ge (

)

2

4

6

8

chan

ge (

)

OEFMRO2

VeinArtery

VeinArtery

0

20

Rel

ativ

e

-40

-20

0

Rel

ativ

e c

-4

-2

0

2

Rel

ativ

e c

0 40 80 120 160Time (min)

0 40 80 120 16040

Time (min)0 40 80 120 160

4

Time (min)






(day 0)Inflammation

(day 12)Necrosis


150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB



MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)





60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT






















































































v(x) = vmaxtimes[

1 minus(

x minus x0

R

)n] (2)



1010

ms

)(a)

A1 V

(b)A1

(c)

08

09

O2

5

pee

d (

mm

A1 V11

V1

Artery

06

07

sO

10

mm

s)

0Sp

A2 V2 A2Vein

1 2 3 405

Vessel order0

5

Sp

eed

(m

V2

A3

V3

5

10

d (

mm

s)

A3V

(d)

25 ArteryVein)

10

s)

AV4

0Sp

eed V3

15

20Vein

te (

lmin

)

5

pee

d (

mm

A4V4

A4V4 05

10

Flow

rat

0 100 200 300 4000S

p

02 1sO2

1 2 3 400















Skin temperature60

Vessel diameter15

CHb20

(b) )c()a(

V i V i

20

40

e ch

ange

()

5

10

e ch

ange

()

10

15

e ch

ange

()Vein

Artery

VeinArtery

Heating

0 40 80 120 160-20

0

Rel

ativ

e

0 40 80 120 160-5

0

Rel

ativ

e

0 40 80 120 160-5

0

5

Rel

ativ

e

0 40 80 120 160Time (min)

0 40 80 120 160Time (min)

Blood flow speed60

OEF and MRO260

0 40 80 120 160Time (min)

sO28

)f()e()d(

V iV i

40

60

chan

ge (

)

20

40

60

chan

ge (

)

2

4

6

8

chan

ge (

)

OEFMRO2

VeinArtery

VeinArtery

0

20

Rel

ativ

e

-40

-20

0

Rel

ativ

e c

-4

-2

0

2

Rel

ativ

e c

0 40 80 120 160Time (min)

0 40 80 120 16040

Time (min)0 40 80 120 160

4

Time (min)






(day 0)Inflammation

(day 12)Necrosis


150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB



MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)





60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT







































































v(x) = vmaxtimes[

1 minus(

x minus x0

R

)n] (2)



1010

ms

)(a)

A1 V

(b)A1

(c)

08

09

O2

5

pee

d (

mm

A1 V11

V1

Artery

06

07

sO

10

mm

s)

0Sp

A2 V2 A2Vein

1 2 3 405

Vessel order0

5

Sp

eed

(m

V2

A3

V3

5

10

d (

mm

s)

A3V

(d)

25 ArteryVein)

10

s)

AV4

0Sp

eed V3

15

20Vein

te (

lmin

)

5

pee

d (

mm

A4V4

A4V4 05

10

Flow

rat

0 100 200 300 4000S

p

02 1sO2

1 2 3 400















Skin temperature60

Vessel diameter15

CHb20

(b) )c()a(

V i V i

20

40

e ch

ange

()

5

10

e ch

ange

()

10

15

e ch

ange

()Vein

Artery

VeinArtery

Heating

0 40 80 120 160-20

0

Rel

ativ

e

0 40 80 120 160-5

0

Rel

ativ

e

0 40 80 120 160-5

0

5

Rel

ativ

e

0 40 80 120 160Time (min)

0 40 80 120 160Time (min)

Blood flow speed60

OEF and MRO260

0 40 80 120 160Time (min)

sO28

)f()e()d(

V iV i

40

60

chan

ge (

)

20

40

60

chan

ge (

)

2

4

6

8

chan

ge (

)

OEFMRO2

VeinArtery

VeinArtery

0

20

Rel

ativ

e

-40

-20

0

Rel

ativ

e c

-4

-2

0

2

Rel

ativ

e c

0 40 80 120 160Time (min)

0 40 80 120 16040

Time (min)0 40 80 120 160

4

Time (min)






(day 0)Inflammation

(day 12)Necrosis


150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB



MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)





60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT











































































Skin temperature60

Vessel diameter15

CHb20

(b) )c()a(

V i V i

20

40

e ch

ange

()

5

10

e ch

ange

()

10

15

e ch

ange

()Vein

Artery

VeinArtery

Heating

0 40 80 120 160-20

0

Rel

ativ

e

0 40 80 120 160-5

0

Rel

ativ

e

0 40 80 120 160-5

0

5

Rel

ativ

e

0 40 80 120 160Time (min)

0 40 80 120 160Time (min)

Blood flow speed60

OEF and MRO260

0 40 80 120 160Time (min)

sO28

)f()e()d(

V iV i

40

60

chan

ge (

)

20

40

60

chan

ge (

)

2

4

6

8

chan

ge (

)

OEFMRO2

VeinArtery

VeinArtery

0

20

Rel

ativ

e

-40

-20

0

Rel

ativ

e c

-4

-2

0

2

Rel

ativ

e c

0 40 80 120 160Time (min)

0 40 80 120 16040

Time (min)0 40 80 120 160

4

Time (min)






(day 0)Inflammation

(day 12)Necrosis


150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB



MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)





60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT



































































(day 0)Inflammation

(day 12)Necrosis


150150

b(g

l)

0

CH

b

(b)1

O2

0 4

sO

60

80

ow

rat

e 15

OE

F

15

MR

O2

(c)04

TreatedControl

20

40

No

rmal

ized

flo

05

10

No

rmal

ized

O

05

10

No

rmal

ized

M

00B 0 3 6 9 12 15 30

Time (days)

N

00B 0 3 6 9 12 15 30

Time (days)

00B 0 3 6 9 12 15 30

Time (days)

N

BL LBLB



MT

1 0

20

lized

flo

w r

ate

(b)VD

00

10

No

rma

15

F

MT

VD

05

10

orm

aliz

ed O

EF

MT MT

14

16

MR

O2

00

No

08

10

12

No

rmal

ized

M

050 z (mm)0 3 7

06

Time (days)





60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT


































































60 210 170 1 0(b) )d()c()a(

50

60

ht (m

g)

190

210

met

er (

m)

160

170

ter

bloo

d)

09

10

2

30

40E

ar w

eigh

170

Ves

sel d

iam

140

150

Hb (g

ram

lit

07

08

VeinArtery

sO2

0 10 2020

Time (days)0 10 20

150

Time (days)

V

0 10 20130

Time (days)

CH

0 10 2006

Vein

Time (days)

6

8

min

)

04 016

lmin

)

050

055

min

)

(h)

4

6

w r

ate

(l m

02

03

OE

F

012

014

sum

ptio

n (

040

045

2(m

l100

g

0 10 200

2

Flow

0 10 2001

02

0 10 20010

012

O2

con

s0 10 20

030

035

MR

O2

0 10 20Time (days)

0 0 0Time (days)

0 10 20Time (days)

0 10 20Time (days)

(g)(f)(e)







MT

Day 0

Day 3

C (gl)0 150 0 350C (gl)





(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT


































































(a) (b)

MT

050









(a) (b) (c) (d)

1 0

20

30o

w r

ate

00

10

Flo

12

08

10

OE

F

Day 0 0 yaD0 yaD

06

1 5

20

25

O2

GT

Day 0 Day 705

10

15

MR

O

GT

y y

08

10

sO2Day 7 7 yaD7 yaD

(e)

Extra Intra06

s

050z (mm)

102sO2





(a) 05

m)

GT

z(m

m

(b)0

GT


































































(a) 05

m)

GT

z(m

m

(b)0

GT


































































































































Documents

Label-free oxygen-metabolic photoacoustic microscopy in vivo · thesia, Euthanex). After the experiment, the animal naturally recovered and was returned to its cage. For tumor study,