Compact tissue oximeter based on dual-wavelengthmultichannel time-resolved reflectance

Rinaldo Cubeddu, Antonio Pifferi, Paola Taroni, Alessandro Torricelli, andGianluca Valentini

We developed a compact dual-wavelength multichannel tissue oximeter based on the time-correlatedsingle-photon counting ~TCSPC! technique. The light sources are two pulsed diode lasers ~outputwavelengths of 672 and 818 nm, an average power of 1 mW, a pulse duration of 100 ps, and a pulse-repetition rate as high as 80 MHz!. The time-resolved reflectance photons are detected by a multianodephotomultiplier, and the output signals are redirected by a router to different memory blocks of theTCSPC personal computer board. The system’s accuracy in determining the absorption ma and thereduced-scattering ms9 coefficients and in reconstructing absorber concentrations in diffusive media wastested on phantoms. Preliminary in vivo tissue-oxygenation measurements were performed on healthyvolunteers under different physiological conditions with a minimum acquisition time of 100 ms and aninjected power of less than 100 mW. © 1999 Optical Society of America

OCIS codes: 170.1470, 170.5280, 170.6510, 300.6500.

5

1. Introduction

In recent years light-based spectroscopy and imaging1

have developed from basic laboratory demonstrationsto clinical practice. A better understanding of light–tissue interactions as well as a mathematical descrip-tion of light propagation in tissues has, in fact, allowedone to obtain workable solutions for situations of clin-ical interest. Therefore optical techniques have beenused profitably for the noninvasive assessment andimaging of biological tissues.2–4 Optical ~red andnear-infrared! radiation can, in fact, penetrate deeplyinto tissue, interact with its constituents, and subse-quently be remitted, carrying information that permitsnot only the characterization of tissues with no needfor tissue removal but also the monitoring of physio-logical functions. For example, the absorption of lightby hemoglobin has been used since 1940 to monitortissue oxygen consumption on the basis of the differentspectral features of oxygenated hemoglobin ~HbO2!

The authors are with the Centro di Elettronica Quantistica eStrumentazione Elettronica del Consiglio Nazionale delle Ricercheand Instituto Nazionale per la Fisica della Materia, Dipartimentodi Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32,I-20133 Milan, Italy. R. Cubeddu’s e-mail address is rinaldo.cubeddu@fisi.polimi.it.

Received 9 November 1998; revised manuscript received 26 Feb-ruary 1999.

0003-6935y99y163670-11$15.00y0© 1999 Optical Society of America

3670 APPLIED OPTICS y Vol. 38, No. 16 y 1 June 1999

and deoxygenated hemoglobin ~Hb!. Moreover, thetracking of scattering changes with time has been pro-posed for noninvasively monitoring blood glucose lev-el6,7 and fast signals in the brain cortex.8–10

Let us focus on the problem of tissue ~i.e., muscle,brain! oximetry11; nowadays, commercial optical in-struments that use cw spectroscopy are widely avail-able on the market. Nevertheless, cw systems are notable to measure absolute values of oxygenation. Be-cause they cannot discriminate between the absorp-tion and the scattering contributions in tissue, they areconstrained to the measurement of oxygenationchanges or to the estimation of absolute oxygenationvalues by means of accounting for the effects of scat-tering through a calibration based on data12 from theliterature or by a comparison with estimated data onthe absolute water concentration in tissue.13 More-over, when using the usually accepted method of cali-bration through the differential path-length factor, onehas to take into account that it is wavelengthdependent,14–16 it is a nonlinear function of both theoptical properties,17 and it can differ distinctively fromsubject to subject.18,19 Thus the current goal of devel-oping a tissue oximeter involves the discrimination ofthe absorption and the scattering contributions, possi-bly by means of a simultaneous measurement of bothoptical parameters. This process will eliminate thedependence on data from the literature or calibrations.

Because time- and frequency-domain techniqueshave proved that they can be used to assess simulta-

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neously the optical properties ~absorption and re-duced scattering! of highly diffusive media such ashuman tissue, the development of an instrument forin vivo noninvasive monitoring of physiological func-tions has become a key idea.1,8,9,20–27 Initially, tech-nological and financial constraints somehow limitedthe development of time-domain systems. Now,however, technology has overcome most limits, andcosts have decreased. Thus time-domain instru-mentation is also feasible for portable systems.

In this paper we present the development and thecharacterization of a dual-wavelength, multichannel,time-correlated single-photon counting ~TCSPC! sys-tem for the noninvasive assessment of tissue absorp-tion and reduced scattering. The system’s accuracyin determining the optical parameters was tested onphantoms. Preliminary in vivo results on volun-teers demonstrate the possibility of tracking tissueoxygenation in real time.

2. Materials and Methods

A. System Setup

Figure 1 depicts the experimental setup. The sys-tem employs two pulsed diode lasers ~PicoQuant

mbH, Germany, Model PDL 800! at 672 and 818m, with a pulse duration of approximately 100 ps, aepetition rate of as high as 80 MHz, and an averageower of 1 mW. The outputs of the two pulsed diodeasers are coupled together into a multimode graded-ndex fiber ~OZ Optics, Canada, Model MMF-IRVIS-0y125! by a 50-mm fiber-optic combiner ~OZ Optics,anada, Model FUSEDIRVIS 50y50!. Two fibers ofifferent lengths precede the coupler and ensure thathe pulses at different wavelengths are coupled to-ether without temporal overlap.The two-wavelength signal is then split into two

bers by a fiber-optic splitter ~OZ Optics, Canada,odel FUSEDIRVIS 5y95!. The first fiber receivessmall fraction ~5%! of the power and is fed directly

nto the photomultiplier ~PMT! to account for even-

Fig. 1. Setup of the TCSPC system for dual-wavelength timesynchronization.

ual time drifts of the instrumentation and to providetime reference. The other fiber receives most of

he power and delivers light to the sample. The re-itted light is collected by a 1-mm plastic–glass fiber

Quartz et Silice, France, Model PCS1000W! in re-ectance geometry.The time-resolved reflectance curves are detected

y a metal-channel dynode PMT ~Hamamatsu, Ja-an, Model RS5600U-L16! and measured by a TP-PC PC board ~Becker and Hickl GmbH, Germany,odel SPC-300! with an acquisition frequency as

igh as 1 MHz and a temporal resolution of 25 ps.The system can also work in a multiple-injection

nd a multiple-collection configuration. A broad-and fiber-optic splitter ~OZ Optics, Canada, ModelUSEDIRVIS 25y25y25y25! can be inserted to pro-ide four independent injection fibers. When asany as four collection fibers are used a four-

hannel router ~Becker and Hickl GmbH, Germany,odel HRT-41! that is equipped with 16 indepen-

ent anodes redirects the output signals of the PMTo different memory locations on the TCSPC PCoard.A homemade software program that is written in

NSI C and is based on the LabWindowsyCVI devel-pment environment ~National Instruments Corpo-ation, Texas! controls data acquisition and analysis.

The typical instrument response functions that arebtained from the injection fiber and the collectionber have a FWHM of approximately 200 ps at bothavelengths. Figure 2 shows typical experimental

urves that were obtained with our system. Notehat the two wavelengths are injected simultaneouslynto the sample and that time-resolved reflectanceurves are thus acquired simultaneously from theample at the two wavelengths without the need forwitching the two laser heads on and off. Moreover,hen the system runs in the multiple-injection–ultiple-collection mode all the curves are acquired

imultaneously.

lved reflectance measurements. PMT, photomultiplier; SYNC,

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B. Tissue Phantom

For the in vitro calibration measurements a cylindri-cal glass box with an 8-cm diameter and an 8-cmheight was filled with an aqueous solution of In-tralipid ~Pharmacia, Italy! to mimic a scattering tis-sue. Subsequently, quantities of ink @Rotring,Germany: Numbers R591-017 ~black!, R591-009~blue!, and R591-007 ~green!# were added to the In-tralipid solution to simulate the presence of differentchromophores in the tissue. Throughout this paper,the concentration of Intralipid refers to the fraction ofsolids in the examined solution, whereas the concen-tration of ink pertains to the fraction of the originalproduct, as obtained from the supplier, present in thesolution under study.

The scattering properties of Intralipid and the ab-sorbing properties of the inks were measured previ-ously by use of time-resolved reflectance spectroscopyunder experimental conditions ~an interfiber distanceof 2 cm, a count number of 105, ma , 0.1 cm21, ms9 .10 cm21! that can safely be applied to our theoretical

odel, thus providing reliable results that are com-arable with those obtained through independentechniques.27 This reliability allowed us to fix thebsorbing and the scattering properties of the phan-om solutions to values typical for human tissues inhe visible and the near-infrared regions of the spec-rum.

The injection and the collection fibers were placedt a relative distance of r 5 1.0–2.0 cm from eachther, in contact with and at right angles to the sam-le surface. A black rigid-plastic strip held the fi-ers firmly and prevented possible direct ~notemitted! stray light from being collected by the fiber.

C. Algorithms

If only two nonreacting substances of concentrationsC1 and C2 contribute to the total absorption coeffi-cient ma of a sample and if their extinction coefficientse1 and e2, respectively, are known at two different

Fig. 2. Typical time-resolved reflectance curves and referencepulses. Note, in each reference pulse, the presence of the charac-teristic afterpulse of the PMT. The arrows mark a FWHM of 200ps.

672 APPLIED OPTICS y Vol. 38, No. 16 y 1 June 1999

wavelengths l1 and l2, respectively, then two inde-pendent measurements of ma at these same two wave-lengths allow one to determine the concentrations C1and C2 on inversion of the Beer–Lambert law:

C1 5ma

l1e2l2 2 ma

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C2 5ma

l2e1l1 2 ma

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In the case of tissue-oxygenation measurements thegoal is to obtain absolute estimates of the HbO2 andthe Hb content, @HbO2# and @Hb#, respectively, to de-

uce the oxygen-saturation fraction, Y 5 @HbO2#y@HbO2# 1 @Hb#%, and the total hemoglobin con-

centration, THC 5 @HbO2# 1 @Hb#. If we assumethat HbO2 and Hb are the only constituents thatcontribute to absorption their concentrations can beestimated by use of Eqs. ~1!, where the extinctioncoefficients at the two wavelengths are derived fromthe literature.28–30

In the time domain, according to diffusion theory,the probability of detecting a photon on the surface ofa turbid semi-infinite medium at a distance r fromthe injection point after a time t can be expressed as31

R~r, t! 512

~4pvD!23y2t25y2 exp~2ma vt!expS 2r2

4DvtD3 Hz0 expS2

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where v is the speed of light in the medium, z0@5~ms9!

21# is the effective mean-free path, D ~5z0y3!is the diffusion coefficient, ze @52D~1 1 rd!y~1 2 rd!#is the extrapolated distance, and rd can be approxi-mated by rd 5 21.440n22 1 0.710n21 1 0.668 10.0636n, with indices of refraction of n 5 1.33 for theexperiments on phantoms and n 5 1.4 for the in vivomeasurements.

Equation ~2! is convoluted with the reference pulse,used as the instrument response function to take intoaccount both time drifts and shape changes, and thenfitted to the experimental data. The best fit is ob-tained by minimization ~the Levenberg–Marquardalgorithm32! of the reduced x2 while both ma and ms9are varied. The range of the fit includes all the ex-perimental points and a number of counts that arehigher than 80% of the peak value on the leadingedge of the curve and than 1% on the trailing edge.33

These counts correspond to approximately 100 datapoints. The use of only two free-fitting parametersand the limited number of data points to be fittedallow us to achieve fitting times of approximately 100ms on a PC ~an Intel Pentium 166-MHz processor!,resulting in on-line data analysis.

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3. Experiments

A. Tests on Phantoms: Single Collection

A first experiment was designed to test the system’saccuracy in assessing the optical properties of a dif-fusive medium. In this experiment, we used asingle-collection reflectance geometry with the injec-tion fiber and the collection fiber placed at a distanceof r 5 2.0 cm relative to each other. The absorptionand the reduced-scattering coefficients were varied inthe ranges 0–0.5 cm21 and 6–24 cm21, respectively.In the first test, we fixed the Intralipid concentrationat 7.7 3 1023 M and varied the black ink contentrom 0.9 3 1025 M to 7.1 3 1025 M. In the secondest, we fixed the ink concentration ~1.9 3 1025 M!

and increased the Intralipid content from 3.8 3 1023

M to 17.9 3 1023 M. At each concentration, thecquisition time was fixed at 3 s, and approximately.5 3 105 counts were collected by proper variation of

the incident laser power.The system proved able to measure the absorption

Fig. 3. Single collection measurements at 672 nm ~filled diamond~b! the reduced-scattering coefficient ms9 as functions of the ink cscattering coefficient ms9 as functions of the Intralipid concentrati

coefficient ma, as illustrated in Fig. 3~a!. At bothavelengths, we observe a linear increase in the ab-

orption coefficient as a function of the ink concen-ration. The dashed lines in the plots representinear least-squares fits to the absorption coefficienta values. As expected, the relative difference be-

tween the slopes of the two lines ~i.e., the specificbsorption coefficients at 672 and at 818 nm! is less

than 3%. In fact, the black ink is characterized by aflat spectrum in the visible and the near-infraredregions; thus the values of the absorption coefficientat 672 and 818 nm are quite similar. Table 1 showsthat the estimates of the absorbance that were ob-tained with our system are in good agreement withthe results achieved with independent techniques.27

The error is less than 6% at both wavelengths, andthis result reveals the good accuracy of this time-resolved reflectance system. From Fig. 3~b!, we

ote that the reduced-scattering coefficient isather insensitive to the ink concentration ~i.e., tobsorption variations! and is lower at the longer

d 818 nm ~open diamonds!: ~a! The absorption coefficient ma andtrations. ~c! The absorption coefficient ma and ~d! the reduced-

s! anoncenons.

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Table 1. Extinction Coefficient e of Black Ink at 672 and 818 nm as

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wavelength, as is expected from the spectral fea-tures of Intralipid.34

The system proved able to measure the reduced-scattering coefficient ms9, as shown in Fig. 3~d!. Theddition of a known amount of Intralipid to an ab-orbing solution is revealed by the related incrementf the measured scattering coefficient: ms9 varies

from 7.9 cm21 to 24.0 cm21 at 672 nm and from 6.5m21 to 17.9 cm21 at 818 nm. The dashed lines inig. 3~d! are the linear least-squares fits to the ms9

values. In agreement with the observations de-scribed above, the slope of the line is higher at 672than at 818 nm. Because of the addition of signifi-cant volumes of Intralipid, the solution is diluted, andconsequently the absorption coefficient shows a weakdecrease as a function of the Intralipid concentration,as displayed in Fig. 3~c!.

The positive intercept in the absorption-linearitylots @Figs. 3~a! and 4~a! ~below!# cannot be explainedompletely in terms of water absorption. This effects due to the higher temporal jitter and the more

arked afterpulse of the compact PMT comparedith the microchannel-plate PMT’s that are usuallysed for time-resolved reflectance measurements.n fact, measurements performed with a double-icrochannel-plate PMT show only a small offset,hich is explained in terms of water absorption. On

he other hand, the positive intercept in thecattering-linearity plot @Fig. 3~d!# can be attributedo the inaccuracy of the theoretical model for low ms9

values, as we showed in previous studies.27 Re-peated measurements on the same sample ~ma 5 0.1m21 and ms9 5 10 cm21! result in fluctuations of lesshan 1% in the measured values of both ma and ms9 if

at least 105 counts are collected.

B. Tests on Phantoms: Multiple Collection

The simultaneous collection of multiple reflectancecurves from different tissue locations might furtherimprove the potential application of a tissue oxime-ter. For example, in the simplest configuration witha single injection fiber and multiple collection fibersat the same distance or at different distances fromthe injection, it is possible to probe different tissuevolumes or depths to obtain spatial maps of the oxy-genation and possibly to discriminate the presence ofeventual subcutaneous fat layers that could nega-tively affect the estimates of oxyhemoglobin and de-oxyhemoglobin.

A second experiment was thus performed with a

Derived from Time-Resolved Reflectance and cw Measurementsa

Measurement Method

Extinction Coefficient@103 cm21y~mlinkymltot!#

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Time-resolved reflectance 5.7 5.6cw 6.0 5.9

aThe cw measurements are described in Ref. 20.

674 APPLIED OPTICS y Vol. 38, No. 16 y 1 June 1999

multiple-collection reflectance geometry to test themeasurements’ reproducibility among independentacquisition channels. Two pairs of identical collec-tion fibers were placed symmetrically with respect tothe injection fiber at distances of 1 and 2 cm. Werepeated the absorption-linearity experiment de-scribed in Subsection 3.A ~black ink concentrationsranging from 0.9 3 1025 M to 7.1 3 1025 M and anntralipid concentration of 7.7 3 1023 M, with ancquisition time of 3 s!. A neutral-density filterKodak, Wratten Model No. 96 with an optical den-ity of 1.0! was placed on the PMT cathode oppositehe 1-cm-distance fibers to compensate for the higherumber of photons detected with respect to the 2-cm-istance fibers.Figures 4~a! and 4~b! show the absorption coeffi-

ient and the reduced-scattering coefficient, respec-ively, as functions of the ink concentration at 672m. The results at 818 nm ~data not shown! are

Fig. 4. Multiple collection measurements at 672 nm: ~a! theabsorption coefficient ma and ~b! the reduced-scattering coefficientms9 as functions of the ink concentrations for the four collectionfibers.

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Table 2. Extinction Coefficient e of Black Ink at 672 and 818 nm as Table 3. Extinction Coefficient e of Blue Ink and Green Ink at 672 and

comparable except for lower ms9 values, as expectedfrom the spectral features of Intralipid. The absorp-tion coefficient again is found to increase linearly asa function of the ink concentration. The results aresimilar for the four collection fibers: at any concen-tration the maximum discrepancy is less than 5%.The similarity among the measurements is attestedto by the data in Table 2, in which we report theextinction coefficient e for black ink as derived fromthe least-squares fit to the data. The coefficient ofvariation for eblack is 2.2% at 672 nm and 4.6% at 818

m. The lower sensitivity of the PMT is a possibleause of the noisier data at 818 nm.

The reduced-scattering coefficient again is found toe rather constant as a function of ink concentration,ut the reproducibility is lower, and, moreover, theres an overestimation of approximately 20% with thebers at the shorter distance from the injection loca-ion. This trend has to be taken into account whennterpreting the time-resolved reflectance data withhe diffusion theory in these experimental conditions,s previously discussed in Ref. 33.

C. Tests on Phantoms: Absorbers’ Concentrations

A further set of experiments on phantoms was de-signed to test the performance of our system in de-termining the absolute concentrations of twoabsorbers in a diffusive medium. As discussed inSubsection 2.C, when considering a medium made oftwo different absorbers the concentrations of eachsubstance can be determined through Beer’s law ifthe spectral features of the specific absorption areknown in advance and independent measurementsare performed at two different wavelengths.

We thus estimated the specific absorption at thetwo test wavelengths for blue ink and green ink,which were chosen to mimic tissue chromophoressuch as oxyhemoglobin and deoxyhemoglobin. Weobtained the estimates by performing an absorption-linearity experiment and fitting the data to a straightline for the measured absorption coefficient as a func-tion of the ink concentration.

Table 3 reports the values of the extinction coeffi-cients at 672 and 818 nm for blue ink and green ink.Both inks show great differences in the extinctioncoefficients at 672 nm compared with those at 818nm. Moreover, the extinction coefficient of the blueink at 818 nm is several times lower than the extinc-

Derived from Time-Resolved Reflectance Measurements for the Case ofMultiple Acquisitions

Fiber Distance ~cm!

Extinction Coefficient e@103 cm21y~mlinkymltot!#

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1A 1 5.8 6.01B 1 5.7 5.62A 2 5.6 5.42B 2 5.6 5.5

tion coefficient of the green ink, and similar valueswere obtained at 672 nm.

Next four solutions were prepared by use of thesame Intralipid concentration ~7.7 3 1023 M! anddifferent amounts of blue ink and green ink ~rangingrom 1.9 3 1025 M to 13.3 3 1025 M!. As summa-

rized in Table 4, solution A contains 100% green ink,solution B contains 100% blue ink, solution C hasequal volumes of blue and green inks, and solution Dcontains blue and green ink volumes in a proportionof 1:2, respectively. The solutions were measured inreflectance geometry with r 5 2.0 cm. For eachalue of the ink content approximately 2.5 3 105

counts were collected with an acquisition time of 3 sby proper variation of the incident laser power. Theundesired offset in the measured absorption coeffi-cient ma ~see Figs. 3 and 4! was subtracted beforevaluating the concentrations.Figure 5~a! shows the measured @i.e., obtained with

qs. ~1!# green ink and blue ink concentrations asunctions of the expected ink concentration for solu-ion A. The dashed straight lines represent the ex-ected concentrations for the two inks, that is, thenity line for green ink and the zero-slope line forlue ink. Both concentrations are reconstructedith good accuracy and precision. The average er-

or for green ink is 3.5%. Figure 5~b! shows theeconstructed concentrations for solution B. Again,he accuracy and the precision are good. The aver-ge error for blue ink is 2.9%. Because solution And solution B contain only one chromophore, a sin-le wavelength measurement of the absorption coef-cient is sufficient to derive its absoluteoncentration. Nevertheless, the good results ob-ained by Eqs. ~1! ensure the robustness of theethod.Figures 5~c! and 5~d! show the reconstructed ink

oncentrations for solutions C and D, respectively.ll combinations of green and blue ink are resolved

818 nm

Ink

Extinction Coefficient e@103 cm21y~mlinkymltot!#

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Blue 2.86 0.10Green 2.78 0.63

Table 4. Ink Composition of the Solutions Under Test

Solution

Ink ~%!

Green Blue

A 100 0B 0 100C 50 50D 66.7 33.3

1 June 1999 y Vol. 38, No. 16 y APPLIED OPTICS 3675

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successfully with good precision. The maximum ab-solute error for the green ink ~blue ink! is 6.8%11.8%! for solution C and 5.3% ~8.1%! for solution D.ome concentrations are characterized with lower ac-uracy, but the total ink concentration is always as-essed with good accuracy and precision. Thisccuracy is important, for example, in the case ofissue-oxygenation measurements for which the totalemoglobin content has to be assessed.

D. In Vivo Experiment: Cuff Occlusion of the Arm

Five adult volunteers ~four males and one female,ith ages ranging from 25 to 35 years! were recruited

rom the laboratory, and informed consent was ob-ained from each subject. Measurements were per-ormed on the medial aspect of the right arm, awayrom any palpable bone.

For time-resolved reflectance measurements thenjection and the collection fibers were held normallyo the skin and at a distance of r 5 2.0 cm relative to

Fig. 5. Measured versus expected ink concentrations for ~a! solustraight dashed lines represent the total ink, blue ink, and greeconcentration is superimposed on the green ink line and on the b

676 APPLIED OPTICS y Vol. 38, No. 16 y 1 June 1999

each other by a black plastic strip fastened with anelastic strap. The fibers were positioned firmly sothat they generated no pressure on the skin but al-lowed no movement. The arm was wrapped in blackcloth and measurements were performed in a dimroom to decrease the amount of background light.The subject’s arm lay in a comfortable resting posi-tion on a flat surface. A pneumatic cuff was placedloosely around the arm. A standard protocol wasfollowed. After an initial period ~3 min! in the rest-ing position, the cuff was rapidly inflated to a pres-sure of 250 mm Hg ~250 torr! to provide abruptvascular ~venous and arterial! occlusion. The cuffcclusion was maintained for 5 min with the muscleesting. The cuff was then released, and a recoveryhase followed for 7 min. Time-resolved reflectanceeasurements were performed simultaneously at

wo wavelengths with an acquisition time of 100 ms.o improve the signal-to-noise ratio in the analysisrocedures, we summed 10 curves, leading to an ef-

, ~b! solution B, ~c! solution C, ~d! solution D ~see Table 4!. Thek expected concentrations. Note that the line for the total inkk line in plots ~a! and ~b!, respectively.

tion An in

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fective acquisition time of 1 s. However, whenneeded to follow the dynamics of fast signals, no dataintegration was performed, and single 100-ms curveswere analyzed.

The changes with time of the absorption coefficientand of the reduced-scattering coefficient at the twowavelengths in one of the volunteers are displayed inFig. 6. In the resting period the absorption coeffi-cient at 672 nm @Fig. 6~a!# is rather constant with anaverage value of 0.27 cm21. After cuff occlusion itrapidly increases to 0.49 cm21, and then when thecuff is released it exhibits an undershoot as low as0.23 cm21. It finally rises to a value slightly higher~ma 5 0.30 cm21! than the initial value. On theother hand, the absorption coefficient at 818 nm @Fig.6~b!# immediately after cuff occlusion shows a smallincrease ~from 0.25 to 0.30 cm21!, followed by slowdecrease until an overshoot is produced when the cuffis released. Then the absorption declines to a valueslightly higher than the initial one.

As shown in Figs. 6~c! and 6~d!, at both wave-

Fig. 6. Absorption coefficient ma at ~a! 672 nm and ~b! 818 nm.coefficients are plotted as functions of time during the cuff-occlusiwhen the cuff is inflated ~left-hand line! and deflated ~right-hand

lengths the reduced-scattering coefficient shows asmall decrease during cuff occlusion: from 11 to 10cm21 at 672 nm and from 9.2 to 8.8 cm21 at 818 nm.A return to the initial values is shown when cuff istaken off. The reduced-scattering coefficient ms9changes are almost instantaneous with cuff occlusionand release and clearly are not coupled to the absorp-tion coefficient ma changes, as is evident by a com-parison of Figs. 6~c! and 6~d! with Figs. 6~a! and 6~b!.Moreover, as expected in the case of scattering, thetrend is similar at both wavelengths.

In Fig. 7 the hemoglobin saturation Y and the THCre displayed, as derived through Eqs. ~1! by use ofata from the literature for the extinction coefficientsf oxyhemoglobin and deoxyhemoglobin.21 As was

already determined for the data collected from phan-toms, the constant offset was subtracted from themeasured ma to allow a more precise evaluation of Yand THC.

During the resting period the hemoglobin satura-tion is rather constant at approximately 72%. After

ced-scattering coefficient ms9 at ~c! 672 nm and ~d! 818 nm. Theperiment. The two vertical lines in each plot indicate the times

.

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cuff occlusion, the increase in deoxyhemoglobin gen-erates a marked reduction of the hemoglobin satura-tion to approximately 45%, whereas cuff releasecauses a fast overshoot to 85% and a slower return toinitial values.

The THC presents smaller variations: It in-creases from 0.23mM to 0.29mM when the cuff isnflated; it then remains rather constant while theuff is on, and finally when the cuff is deflated ithows an overshoot to 0.34mM, which decays rapidlyo approximately 0.26mM, a higher value than thenitial one at least in the first 6 min.

The 250-mm Hg cuff inflation determines venousnd arterial occlusion, i.e., no oxygenated blood isupplied to the muscle and no deoxygenated bloodows away, but at the same time the muscle burnsxygen. Thus we expect a decrease in Y withoutajor changes in the THC, as were obtained experi-entally. Note also the burst in the THC and Yhen cuff release permits the flow of new oxygenatedlood to the region under test.

Fig. 7. ~a! Hemoglobin saturation Y and ~b! the THC as functionsof time during the cuff-occlusion experiment. The two verticallines indicate the times when the cuff is inflated ~left-hand line!and deflated ~right-hand line!.

678 APPLIED OPTICS y Vol. 38, No. 16 y 1 June 1999

Water absorption is negligible at 672 nm, and itakes only a minor contribution to the total absorp-

ion measured at 818 nm. Moreover, its effect andhose of all other chromophores leading to a constantbsorption are eliminated by subtraction of the offsetrom the measured absorption. Myoglobin canardly be discriminated from hemoglobin because ofhe spectral overlap, but the hemoglobin response isrimary in aerobic and ischemic studies35; thus mea-

sured changes in Y and the THC are expected to beue primarily to hemoglobin.As was described in Subsection 3.C, good precision

s achieved in the assessment of the total concentra-ion of the present chromophores. Similarly, theeasure of the THC is expected to be performed re-

iably. With respect to Y, the estimated error is ofthe order of the error obtained in the evaluation of@HbO2#, i.e., less than 12%, with reference to the datareported in Subsection 3.C.

In Fig. 8 we again consider the hemoglobin satu-ration Y in the time period following the instant whenthe cuff is deflated. Hemoglobin saturation Y isvaluated from single curves obtained with acquisi-ion times of 100 ms instead of integrating 10 curves.uch a high sampling rate is necessary because theime scale of the phenomenon under study is veryhort, as can be seen from Fig. 7~a!. By an exponen-ial fit, we can derive the time constant that is char-cteristic of the fast increase in hemoglobinaturation Y. In the case under study recovery is

characterized by a time constant of approximately4.7 s. Note that, because of the limited acquisitiontime, each time-resolved curve has approximately 104

total counts with an injected power of less than 100mW ~corresponding to an energy of less than 10 mJ!,nd this lower energy results in a higher variation inhe fitted optical properties and thus in Y values.

Fig. 8. Hemoglobin saturation Y during recovery after cuff defla-ion. The acquisition time is 100 msypoint with an injected power

of ,100 mW. The vertical line indicates the time when the cuff iseflated.

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Nonetheless, the possibility of acquiring data at sucha fast sampling rate permits not only sensitivity tofast physiological signals but the elimination of arti-facts caused by rapid sources of noise ~e.g., pulsation,espiration, or muscle contraction!, thus further ex-ending the potential applications of the system.

4. Conclusions

A basic requirement for an instrument dedicated toquantitative measurements of absorbers in highlydiffusive media like human tissues is discriminationbetween the absorption coefficient and the reduced-scattering coefficient. Moreover, for practical usethe system must be accurate, portable, fast, robust,and possibly cheap.

Instruments for cw spectroscopy have been devel-oped and used widely in clinical practice, but they donot allow absolute measurements. Frequency-domain systems and time-resolved systems are bothable to assess ma and ms9. As a result of financial andtechnical constraints, up to now frequency-domain,near-infrared spectroscopy instruments have beenmore competitive than time-domain instruments.Currently available time-resolved systems20–23 havethe disadvantage of a long acquisition time ~20–30 so obtain a suitable signal-to-noise ratio!. Nonethe-ess, they have been demonstrated to be robust, ac-urate, and flexible.

The system we have proposed is portable, thanks tohe use of diode lasers and a compact dynode PMTith a compact 2900-V power supply instead of aulky argon-pumped dye laser, an expensiveicrochannel-plate PMT, and a higher-voltage

23000-V! power supply. A standard PC board issubstituted for the Nuclear Instruments modules of atypical electronic chain for TCSPC measurements.Moreover, a synchronization signal from the laserdriver effectively replaces the output signal of a fastphotodiode to provide a reference pulse for the TC-SPC board. Other important elements for portabil-ity are fiber-optic cables, splitters, and couplers,which must be chosen carefully to minimize powerlosses. The system is fast. The diode lasers oper-ate at a repetition rate as high as 80 MHz, whereasthe TCSPC board has an acquisition frequency of 1MHz. Good sensitivity is achieved. In fact, undertypical in vivo conditions a measure with a reason-ble signal-to-noise ratio is obtained with a minimumcquisition time of 100 ms and an injected power ofess than 100 mW. The system is flexible: detach-ble laser heads permit simple wavelength selection.oreover, the multianode PMT and the four-channel

outer together with the fused splitters–couplers of-er both single and multiple injection and collectiononfigurations. Finally, the system is reasonably in-xpensive: the total cost of the separate elements ispproximately $30,000.00.In conclusion, we have demonstrated the feasibility

f tissue-oxygenation measurements by using a por-able time-resolved instrument. With tests onhantoms the system has been characterized inerms of accuracy and precision in the determination

f the absorption coefficient and the reduced-cattering coefficient and in the estimate of the abso-ute concentration of two different absorbers within aighly diffusive medium. Preliminary in vivo mea-urements of tissue oxygenation under differenthysiological conditions were performed on healthyolunteers, confirming that the system is sensitive tohanges in the functional parameters.

Nevertheless, there are currently some constraintshat are mainly due to the nature of biological tissue.n vivo measurements are affected by the heteroge-eity of the sample: layered media ~fat layers!, lo-alized absorbers ~capillaries and blood vessels!, andpurious background absorption because of the pres-nce of other chromophores besides hemoglobin ~wa-er, cytochrome-aa3, myoglobin, melanin, lipids!

should be taken into account.Future study and development of the system

should be directed toward an increase in the numberof wavelengths and the use of multiple injection andcollection measurements to map tissue oxygenationand thus overcome the nonuniformity of the sampleor to test different regions simultaneously.

References and Notes1. B. Chance and A. Yodh, “Spectroscopy and imaging with dif-

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2. Special issue on Diffusing Photons in Turbid Media, Appl. Opt.36~1!, ~1997!.

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1

1

1

1

1

1

1

1

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3

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