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Proc. Natl. Acad. Sci. USAVol. 90, pp. 3423-3427, April 1993Medical Sciences

Highly sensitive object location in tissue models with linearin-phase and anti-phase multi-element optical arrays inone and two dimensionsB. CHANCE*t, K. KANG*, L. HE*, J. WENG*, AND E. SEVICK**Department of Biochemistry and Biophysics, Johnson Foundation, University of Pennsylvania, Philadelphia, PA 19104-6089; and tDepartment of ChemicalEngineering, Vanderbilt University, Nashville, TN 37325

Contributed by B. Chance, December 4, 1990

ABSTRACT Based upon previous observations of low-frequency photon diffusion waves within highly scatteringtissue, this paper explores the "near-field" phenomena of suchwaves of 10-cm wavelength with 200-MHz phase modulationequipment. Multiple-element source arrays consist of laserdiode sources modulated at 1800 out ofphase with respect to theother sources. The diffusing waves originating from the out-of-phase sources give, in the midplane, an amplitude null anda sharp phase transition. These may be observed in a highlyscattering intralipid medium simulating the breast or brain(0.5% intralipid), 3-5 cm from the transmitting laser diodes.In the plane containing the array, there is a high sensitivity fora small volume of a hidden absorber (indocyanine green) deepwithin a highly scattering medium; 20 pmol in a volume of 70,ul can be detected. Two-dimensional arrays consisting of fouror more elements in two orthogonal planes give sensitivity onboth axes similar to the one-dimensional array. Measurementsshow that in the presence of a light-absorbing object, theamplitude null and the interference plane becomes a curvedsurface which is deflected toward the heterogeneity. The degreeof deflection is related to the volume and the absorptioncharacteristics of the heterogeneity and provides detection ofthe heterogeneity, and thereby may provide localization infor-mation for the detection of small tumors within the humanbreast, or stroke volumes, aneurysms, and tumors in thehuman brain.

Approximately a half-dozen approaches are under study invarious laboratories for imaging under conditions of highlyscattered light (14). The apparently simplest method is that ofballistic or snake imaging, where the scattered rays arediscarded and only those whose pathlength approximates thegeometric input/output distance are accepted (1-5). Selec-tion of these pathways is achieved by time gating or phasedcoherence at the optical frequency. Such methods, whileemploying traditional computer tomography image recon-struction algorithms and thereby affording rapid data acqui-sition, are limited either in sensitivity or in tissue penetrationto small organs that are of less importance than breast andbrain. Furthermore, the desirable laser power levels mayexceed current regulations.

Imaging processes that involve the entire time course ofphoton migration are under study by Wilson et al. (6) andDelpy et al. (7). This method, which is powerful from thestandpoint of signal/noise ratio, suffers from the fact thatmany data points are required to afford "over-stuffed"matrices and require at the same time a sophisticated imagereconstruction algorithm, direct or indirect.Two-dimensional images are well defined and progress

toward three-dimensional reconstruction is being made, par-

ticularly in multi-pixel approaches where an image intensifi-er-CCD (charge-coupled device) camera coupling is gainmodulated at high frequencies, affording a two-dimensionalphase and amplitude modulation images (8). The drawback tothis approach is that "difference" or "ratioing" images mustbe used for detection and localization. The "absence" con-dition for data acquisition may be satisfied by scans ofnormaltissue volumes adjacent to the tumor-containing volume.The achievement of sharpened or focused photon density

patterns in tissue was suggested by Gratton and coworkers (9and §) and by Knutson and coworkers (10), and the opticalanalogies have been explored by Yodh and coworkers (11).By use of in-phase and out-of-phase light source arraysmodulated at frequencies near 200 MHz, highly directionalamplitude minimal (nulls) and sharp phase transitions areobserved in scatterers that mimic the brain or breast. Thispaper reports theory and experiments in near-field in-phase/anti-phase optical arrays. By using a four-element [twoin-phase (<00) and two anti-phase (<1800) light sources], wereport here highly significant localization (signal/noise > 5)of a 60- to 300-/Al volume containing 20 pmol of an absorberin a 3-mm diameter tube obscured deep within highly scat-tering material.

Theory

Because photon diffusion waves are propagated in the scat-tering medium and maintain phase coherence for at least awavelength (shown in Fig. 1 to be -10 cm at 200 MHz; ref.12), phase encoding of various portions of the optical fieldshould afford sharp transitions of phase when a detector istraversed from one phased lobe to the other, or indeed whenthe relative phase of the lobes is altered for a fixed detectorposition. This communication evaluates the sensitivity ofphase and amplitude transitions for absorber detection inhighly scattering media.Phase modulation or phase shift spectroscopy measures

transport delay in terms of phase shifts. The phase delay, 0(in radians), between the source and the detector may beexpressed as

P PdO(p) = 2ir-= 2ir-= 2lTrf P

Aw T VW' [1]

where Aw is the wavelength, Td is the transport delay, T is theperiod of the sinusoidal wave, and f is the frequency of thewave.When Eq. 1 is expressed for the velocity of the wave, Vw,

tTo whom reprint requests should be addressed.§Gratton, E., Mantulin, W. W., van deVen, M. J., Fishkin, J. B.,Maris, M. & Chance, B., Proceedings of the Third InternationalConference on Peace Through Mind/Brain Science, Aug. 5-10,1990, Hamamatsu, Japan, pp. 183-189.

3423

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

3424 Medical Sciences: Chance et al.

pVw = 2if 1(. [2]

e(p)

For a system which has no boundary, the phase shift, 0(r)linf,can be expressed as

O(P)Iinf =

-pr [3(ga + g4)][1+2

\]}r sin -,

I

s

JaC 2

[31

where

= tani(±ainI) [4]

p and 1 are the absorption and the effective scatteringcoefficient, respectively; the diffusion coefficient D =1/[3(p + A')]; Cn is light velocity in the scattering medium,which is c/n (c is light velocity in vacuum and n is therefractive index of the medium); and the subscript "inf"denotes infinite space domain.

Therefore, for an infinite domain,

[12irf2] -1/2 -1/2Awl1.=2r{ [a1 + - sin- [5]'WLff D [\ aCn/ 2

Fig. 1 shows wavelength for a variety of absorption (p)and scattering (1') values within the physiological range atoscillation frequencies from 107 to 109 Hz. A frequency of200MHz affords a wavelength of -10 cm for most physiologicalconditions, whereas the lower frequencies give values ofwavelength highly dependent upon small values of a andupon larger values of 4..

Method

The single-frequency phase modulation system operating at200 MHz is illustrated in Fig. 2A. In principle, two identicaloscillators establish modulation of the light source and of thephotomultiplier detector with a frequency difference of25 kHzas a subcarrier that contains the essential phase and amplitudeinformation. A "push-pull" transformer provides in-phase(<0°) and anti-phase (<1800) drive for the two pairs of laserdiodes operating at 780 nm. Fig. 2B illustrates the transmitter/receiver geometry for a linear array ofin-phase and anti-phaselaser diodes operating at 780 nm with a power of 7 ,uW and amodulation of 90%. The array and detector are immersed inan "infinite" fish tank filled with 0.5% intralipid, K4 10

cm-l, p < 0.02 cm-'. The receiver can be moved withrespect to the array in the absence ofan intervening absorber.

A250

200

150

-^ 100

so,

P,, cm-,+ 0.01

--0-0.03

-u0.05-*-3*0.07- - - 0.09

B250

200

6150d: 100

so

.....107 1~08

Frequency

14. cur'

510

-.--20- - - 25

108

Frequency

FIG. 1. Wavelength changes with changes in the physiologicalrange of absorption coefficient and scattering coefficients. (A) Wave-length change with the change in absorption coefficient at constantscattering coefficient, 10 cm-', (B) Wavelength change with thechange in scattering coefficient at constant absorption coefficient,0.05 cm-' in Naperian logarithms.

FIG. 2. (A) Electronic circuit for coupling ofa four-element laserdiode linear array originating at 780 nm in which adjacent pairs are0 phase and 1800 phase. The phased array is driven at 200 MHz witha local oscillated frequency of 200.025 MHz. Phase and ac amplitudeoutputs are indicated. (B) Geometry of the four-element array inwhich two of the laser diodes are at 0 phase (S1, S2) and two are at180° phase (S3, S4). The spacing is usually 3.5 cm. The detector maybe moved parallel to the array and in the plane of the array to locatethe amplitude null and phase transition. The model system containsa background absorber scattered together with a movable absorberscatterer or fluorochrome and a movable detector. The absorber maybe translated between the detector and the transmitter array. Usuallythe detector is at 5 cm and the absorber is midway betweentransmitter and receiver. The sample volume is usually a 3-mm tubeof variable length, usually 40 mm.

Since the in-phase and anti-phase laser diodes are driven bya push-pull circuit with an accurately determined center tap,the amplitudes are well matched. If not, variation in the rfdrive for the individual diodes will normalize the amplitudesto afford a sharp null and an abrupt phase transition asdetermined by scanning the detector within the X,Z planecontaining the array (Fig. 2B).Mapping the Amplitude Nuil and Phase Transition of a

Four-Element Linear Array. The goal of the tests is first toevaluate the position of the amplitude null and phase transi-tion in order to be able to place the detector at the zero pointof the phase transition, which also corresponds to the null inamplitude. The detection fiber is traversed in the plane ofthearray. In the near field, the intensity from the 1800 phase lightsources is nearly constant until the mid-line null is reached.The plots of signal amplitude vs. detector position are thussymmetrical and show a sharp (2-cm full width at half

Ato Transmitter

BDetectorScan

Proc. Natl. Acad. Sci. USA 90 (1993)

-N

Proc. Natl. Acad. Sci. USA 90 (1993) 3425

maximum) and deep (20-decibel) minimum of the ac compo-nent of the signal (Fig. 3A).The phase transition from light sources of zero phase to

1800 phase shown in Fig. 3B gives a maximum slope of10°/mm. The phase transition has a number of advantagesover the amplitude transition. The amplitude null extendsover a range of 4 cm, whereas the phase transition is sharper:a shift from 00 to 1800 occurs in a short distance, 2 cm.Further, no phase change is detected until the phase transi-tion is reached and the portion of the array (i.e., left or right)is identified by the value of the phase signal. Thus theambiguities of the amplitude signal are minimized in theone-dimensional linear array.

Perturbation of the Null by an Absorber: Effect of Size of theAbsorber. Once the zero point of phase shift has beendetermined by traversing the detector in the X,Z planecontaining the array, a phantom absorber may be locatedaccording to the diagram of Fig. 2B, midway between trans-mitter and receiver (a distance of 3.5 cm from either one), tomimic a tumor in the breast or brain. The absorber istranslated parallel to the array and in the plane of the arrayto obtain intensity and phase perturbations; values of bothphase and amplitude modulation are noted at each position ofthe absorber (Fig. 4). In this case, the amplitude and phasecharacteristics differ in shape from that noted in the scan ofthe detector, the phase transition takes the form of a reso-nance curve, there is zero perturbation of phase at distanceson the X axis >5 cm from the phase transition of a four-element array, and phase shifts in the positive or negativedirection as the phase null are observed. As a criterion ofsensitivity, we take again the steepest slope of the phasetransition perturbation (12°/mm for the 1.5-mm diameterobjects, 0.05 mm).

Fig. 5 shows the response to long cylindrical objects (>70mm; see Fig. 8) of various diameters. The data show an initialslope of 100 phase shift per mm of phantom diameter with asaturation level at 5-mm diameter. With a phase detectabilityof 0. 10, a motion of 0.01 mm is at the noise level of detection.If a hidden absorber is to be imaged by rotation of thetransmitter/receiver around a large cylindrical object (breastor head), the error of angular localization of a 3-mm-diameterobject at a depth of 3.5 cm is <<10 at the noise level.

Effect of Absorber Concentration. Fig. 6 illustrates theperturbation of the phase transitions obtained at 780 nm bytranslating a "black" rod (o), two concentrations (70 ,ug/ml,o; 3.5 ,g/ml, *) of a near-IR-absorbing dye (Ama = 805 nm;E = 200 cm-1mM-1; Mr = 770, indocyanine green), allfreshly dissolved in 0.5% intralipid and intralipid only (O) ina 3-mm-diameter thin-walled glass container 11 mm long.

A> 80'

° 60x

-o 240

*-- 20

0 o

B200

uz

100

'0, 0o

sPL4

4 8 12 16 0 4 8 12Detector position inX axis, cm

16

FIG. 3. Amplitude (A) and phase (B) data for element array of Fig.2. The source positions, S, are indicated in Fig. 2B. (A) Amplitudecharacteristic of the array of Fig. 1 where the midpoint of the arrayis at 7.5 cm on the abscissa. The intensity null is at least 40 decibels,obtained by translating the detector parallel to the array in which theintensities are matched and phases are 1800. (B) Phase transitions forelement array of Fig. 2 differ by translating the detector parallel tothe array as in Fig. 2A. The phase shift of 1800 is obtained within aspan of 2 cm, the rate of change of phase is 10°/mm of displacementof the detector.

A

I

'0

aa:

B

P IW ' T.. I * , .

-64 -2 0 2-4 6 8 68 44 -2 0 2 4 6Absorber Position in X-axis (cm)

FIG. 4. Intensity (amplitude) and phase plots for perturbation ofthe amplitude null and phase transition of a four-element array witha detector-transmitter spacing of 5 cm in the arrangement of Fig. 2B.(A) Perturbation of the amplitude null for a cylinder of 1.25-mm-diameter (o). (B) Perturbation of the phase transition for a 1.25-mm-diameter black rod 7.0 cm long, 3.5 cm deep within 0.5% intralipid(o).

With indocyanine green at 3.5 ,ug/ml (U), the slope is 1.60/mm, or a "noise level" of 0.2 ,ug/ml. The volume of thephantom is 0.07 ml and the "noise level" corresponds to 14ng, or 20 pmol.

Fig. 7 shows the profile of phase shift at various absorberconcentrations. The initial slope caused by insertion of theabsorber at the zero point of phase transition corresponds to2°/(,ug/ml). The 0.17-ml volume contains 170 ng, or 220 pmol.At the noise level of phase detection of 0.10, 11 pmol isdetectable. Since only a portion of the 4-cm length of thesample volume may be in the sensitive volume of the opticalfield, we investigate further the effect on the phase pertur-bation of the length of the 3-mm-diameter sample tube filledwith indocyanine green at 17.5 ,ug/ml, as shown in Fig. 8.Apparently, below the 3-cm length the phase perturbation isindependent of length to above 4 cm and decreases linearlywith the sample length thereafter giving a constant sensitiv-ity. The initial slope is 0.70 phase shift per mm length.

Detection of Fluorescent Signals. The excitation emissionproperties of indocyanine green can be measured in wholeblood from the absorption peak at 805 nm and the fluores-cence emission peak at 835 nm extends out to 880 nm. Thisfluorescence is too small to interfere with absorption mea-surements but can be measured with an appropriate guardfilter that attenuates the excitation at 780 nm and transmits>40%o above 850 nm. The detector R928 is sensitive to 850nm; 4-fold better performance would be expected with agallium arsenide cathode detector.

This phenomenon has been studied by O'Leary et al. (11)and the re-emission ofphotons from the hidden absorbers hasbeen shown to produce a new location ofa spherical diffusivewave originating at the object. Object location is possible bydetection of the null planes with in-phase and anti-phaseillumination of the fluorescence object from appropriateorthogonal sites (Fig. 9).

En

(> 40on00So 30-cn 20-0c)CZ,c 10-P0

0 2 4 6aDiameter of the absorber, mm

FIG. 5. Effect of absorber diameter upon the magnitude of thephase transition in 0.5% intralipid.

Medical Sciences: Chance et al.

3426 Medical Sciences: Chance et al.

75

50 ,25

00

-25-

-50-

-750 2 4 6 8 10 12 14

X axis, cm

FIG. 6. Effect of the nature and concentration of absorber onphase transition for element array detector-transmitter separation 5cm, with object location midway between transmitter and detector.Four types of objects are translated between transmitter and receiverand are identified: 5-mm-diameter black rod (e), indocyanine greenat 70 ,ug/ml in 0.5% intralipid (o), indocyanine green at 3.5 ,ug/ml in0.5% intralipid (m), and intralipid only (O). A phase transition of1.5°/mm displacement is obtained in a 3-mm-diameter tube 11 mmlong with indocyanine green at 3.5 ,ug/ml.

Position of the Phase Transition in Two Dimensions. Twoorthogonal phased arrays give a line intersection of theirorthogonal null planes. The scan of the Z-axis array (0) isdisplayed in the null plane X, Y and passes through the pointD, 5 cm from the array. Similarly, a scan of the X-axis array(m) is displayed in the corresponding null plane Y,Z and alsopasses through the point D. The intersection of the two nullplanes X,Y and Y,Z gives a "line of position," Y. A thirdarray, along the Y axis, would give a third profile in the X,Zplane and a three-dimensional location at point D.Motion ofan absorber or a fluorochrome between the array

and pointD in Fig. 9 would give perturbations in the three nullplanes. In the case of a fixed object such as a tumor in thebreast, the null planes are aligned with the object by scanningthe array and the detector with respect to the breast (see Fig.11).

Resolution of Two Objects. The resolution of the phasedarray depends upon a number of geometric parameters andhas been studied by using a two-element array shown in Figs.2B and 9 with an S1-to-S2 distance of 4 cm to determinewhether phase transitions could be observed when twoclosely spaced objects are translated between the transmitterand receiver. Experimentally, translation of two 5-mm-diameter rods, 1 cm apart, shows a 300 phase perturbation inthe 1-cm gap between the rods (180° would be expected witha larger gap). Computer simulation (Fig. 10) of the translationoftwo 1-cm3 cubes in the same geometry shows no phase shiftat 1-cm spacing but 600 at 1.25 cm and increasing to 1700 fora larger spacing of the blocks (see Fig. 10 "second transi-

30-a) 3

20-

10CZ

0uI........................0 20 40 60

Concentration, ,tg/ml

FIG. 7. Concentration dependence of phase shift for inserting a3-mm-diameter, 25-mm-long thin-walled glass tube containing vari-ous concentrations of indocyanine green midway between transmit-ter and receiver separated by 5 cm. The ordinate shows phase changeon interposing the absorber into the phased array. Each concentra-tion is made up in 0.5% intralipid. The initial slope of the phasechange is 2°/(,ug/ml) for the 0.3-ng sample.

a) 20-0

CI 10-

00 40 80 120

Absorber length, mm

FIG. 8. Effect of the length of the 3-mm tube upon the phase shiftobserved on inserting the absorber, indocyanine green (17.5 ,ug/ml)covering a length of the sample tube from 0.7 mm to 140 mm.

tion"). This second transition is better detected ifthe S1-to-S2distance is decreased from 4 cm to 2 cm.Human Breast Scan. To demonstrate that the adipose tissue

of the normal human breast is sufficiently uniform in itsscattering and absorption to give a sharp null, we haveapplied the two-element phased array to the normal adulthuman breast (with appropriate informed consent) and havemechanically scanned the phase transition with the detectoroptical fiber using the geometry of Fig. 2. Fig. 11 shows oneof several scans of the normal human (under 40 years) breastat ==2 cm from the chest. The maximum slope of the phasetransition is 26°/mm, corresponding to an accuracy of loca-tion of the nodal plane in vivo of =10 gm for a phase noiseof 0.30. This would appear to validate the high sensitivities ofstudies of Figs. 4-9 as directly applicable to the human breastfor localization of tumors. Displacement of this null by theincreased absorption and scattering of a breast tumor hasbeen demonstrated.

Discussion

The phase coherence of the low-frequency propagated pho-ton density wave in 0.5% intralipid is maintained over adistance from the source of at least one wavelength of thediffusive wave (7). The "near-field" phenomenon exhibitssharp phase transitions and amplitude nulls for an in-phase/anti-phase linear array. Four-element linear arrays in one ortwo planes give remarkable sensitivity ofthe phase transitionin response to the small amounts ofabsorber (picomole levelsof indocyanine green). This result suggests high sensitivity inthe location of a small absorber or absorber difference withinthe brain or breast.

Yz

y

FIG. 9. Two-dimensional scan of the phase transitions in two nullplanes from two orthogonal in-phase and anti-phase arrays. *, Y,Zplane scan; o, X, Y plane scan. The maximal slope of the two scansis 36°/mm.

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 3427

c

2000-1̂ transition

+ toa.

I0ooo-2nd transition

-to+T

0 o.0 2.0Object Spacing (cm)

FIG. 10. Computer simulation of detection of the aperture be-tween two absorbers. The array of Fig. 2 was used with a 4-cmseparation of in-phase and anti-phase transmitters separated 5 cmfrom the detector. Two 1-cm3 objects at four spacings were translatedparallel to the array and midway between transmitter and receiver.The ordinate indicates the perturbation of the detector phase as thefirst object (m) enters the optical field plane (first transition, + 180° to0°) and the arrival of the second object (o) at the null plane (secondtransition, 00 to 1800).

While this study reports results on linear arrays as trans-mitters, reciprocity of such arrays in transmitter/receivermodes is expected, and higher directionality and sensitivityare expected when both transmission and reception employone- or two-dimensional linear rectangular or multi-elementarrays.

Contrast Agents. It is customary in magnetic resonanceimaging (MRI) and positron emission tomography (PET) tomake an intravenous injection ofcompounds which permeatethe vascular bed in the region of interest and specifically labelthe tissue volume of interest. In the case of PET, thedifferential labeling depends upon the specific biochemicalpathway for the metabolism of the contrast agent. In the caseof MRI, the contrast agents for tumor study depend upon therelative fragility or permeability of the vascular bed servingthe tumor, and in this case the contrast agent is identified bythe potent paramagnetism of rare earths, transition metals,etc.

In optical studies, the contrast agent must rely upon adistinctive absorption or scattering effect. In the case ofabsorbers, the tricarboxycyanine series, of which the bestknown member is indocyanine green (cardiogreen), has beenapproved for use in human liver-function studies for severaldecades and indocyanine green is benign at 50 ,ug/g of bodyweight (manufacturer label, Hynson, Westcott and Dunning,Baltimore). Sensitivities to nanogram amounts of indocya-nine green are demonstrated here in model systems. Theentry of indocyanine green into tumors is expected to involvethe same enhanced permeability as in the case of the MRIprobe. Thus the tumor concentration is expected to be higherthan 50 ,ug/g, while phase array requires only a few micro-grams per gram. In fact, a covalent linkage of the two MRIand optical probes is expected to afford definitive compari-sons between MRI and optical studies.Scanning and Imaging. While the data displayed in this

paper are acquired by step-by-step scanning with a singledetector, multiple detectors in the null plane will give rapidlocation of the null plane. In addition, the null plane may beperturbed by a conical or rectilinear scan, S, by the timevariation of the phase relations of the transmitters to affordprecise localization of hidden objects or the detection ofmultiple objects in tissues (13).

,-WB.:s

0a);n

.-O.

I..0

3

0 10 20 30 40 50Detector Position(mm)

FIG. 11. Illustration of the sensitivity of the determination of theamplitude (o) and phase modulation (0) phase transition in the scanof the normal human breast with 4 cm between in-phase andanti-phase light sources and a compressed normal breast thickness of3.5 cm. The detector fiber was mechanically scanned in incrementsof 0.5 mm across the phase transition. The plane of the array wasnormal and the null plane was parallel to the long axis of the breast.The scan was taken at -2 cm from the chest. The maximum slope is26°/mm. The subject was under 40 years of age.

Summary

A very simple form of hidden-object location in highlyscattering medium resembling breast or brain is based uponmaintenance of coherence of low-frequency photon densitywaves in that medium. This study is based upon the specialcase of multi-element linear arrays in one or two dimensionsbut is readily extended to three dimensions. Phase encodingof constant-amplitude 200-MHz modulation gives high sen-sitivity of angular pointing (10) and in detection of smallobjects (60-300 ,u) containing small amounts (picomolelevels) of an absorber with potential use as an in vivo contrastagent. Improved performance using more elements in trans-mitter arrays with complementary receiver arrays is expectedto afford two- and three-dimensional localization of smallabsorbers/scatterers in brain and breast.

We thank Dr. Arjun Yodh, Ms. Maureen O'Leary, and Mr. DavidBoas for collaboration in this project. This work was partiallysupported by National Institutes of Health Grants CA50766,HL44125, and NS27346 and by American Cancer Society GrantBE-13.

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Medical Sciences: Chance et al.