optical properties of normal and carcinomatous bronchial tissue

Optical properties of normaland carcinomatous bronchial tissue

Jianan Qu, Calum MacAulay, Stephen Lam, and Branko Palcic

To understand better the optical characteristics and autofluorescence properties of normal andcarcinomatous bronchial tissue, we measured the absorption coefficient, scattering coefficient, andanisotropy factor from 400 to 700 nm. We made the measurements by using an integrating sphere witha collimated white-light beam to measure total reflectance and transmittance of samples. Theunscattered transmittance of the samples was measured through polarized on-axis light detection. Theinverse adding-doubling solution was utilized to solve the equation of radiative transfer and to determinethe absorption coefficient and reduced scattering coefficient. The scattering coefficient and anisotropyfactor were derived from the unscattered transmittance of the sample and the reduced scatteringcoefficient. The measured parameters allow us to simulate photon propagation in normal bronchial andtumoral tissue by using Monte Carlo modeling.

Key words: Optical properties, lung tissue, tissue optics.

Introduction

Human tissue is an optically turbid media, and whenphotons propagate through such tissue they arescattered and absorbed by the particles and macromol-ecules inside the tissue. The intrinsic optical proper-ties of tissue determine the reflectance and autofluo-rescence behavior of the tissue. For the progress ofnoninvasive optical detection, photodynamic therapytreatment, and laser surgery of diseased tissues to beenhanced, accurate measurements of the optical prop-erties of human tissue are required. These measure-ments will lead to a better understanding of photon-tissue interaction for the development of more efficienttreatment techniques and more accurate diagnosticdevices.

Most of the recent advances describing photonpropagation in tissue are based on the understandingof radiative transport equations, Monte Carlo simula-tions, or both. Both techniques require knowledgeof the absorption coefficient, [La, scattering coefficient,a,, and the single-scattering phase function for the

The authors are with the Cancer Imaging Department, MedicalPhysics Division, British Columbia Cancer Agency, Vancouver,British Columbia V5Z 1L3, Canada; J. Qu is also with XillixTechnologies Corporation, Suite 200, 2339 Columbia Street, Van-couver, British Columbia V5Y 3Y3, Canada.

Received 10 November 1993; revised manuscript received 30March 1994.

0003-6935/94/317397-09$06.00/0.© 1994 Optical Society of America.

tissue. Research by Jacques et al.l and Yoon et al.2showed that a Henyey-Greenstein function is a goodapproximation for single-particle light scattering.The Henyey-Greenstein function depends on only ananisotropy factor, g, which is equivalent to the aver-age cosine of the scattering angle.

Over the past ten years a variety of techniques formeasuring optical properties of tissues have beenreported, as well as the advantages and disadvantagesof these techniques. 1 2 Cheong et al.'3 provided alist of optical properties for many human and animaltissues. The optical properties measured for sometissues are quite different, which could be attributedto the different experimental methods used in makingthe measurements. In fact, the measurement accu-racy of tissue optical properties strongly depends onthe experimental conditions utilized and on experi-mental postprocessing. The relevant issues are thetissue preparation, experimental setup, theoreticalmodel, and data-correction techniques.

The use of the integrating sphere for total reflec-tance and transmittance measurements of tissue is awell-established technique. Knowledge of total re-flectance and transmittance properties of tissue per-mits the determination of the absorption coefficientand the reduced scattering coefficient, SL,' = (1 - g) 5,of the tissue. Currently, the inverse adding-dou-bling (AD) solution of the radiative transport equa-tion for slab samples when one uses an integratingsphere measurement is a fast and accurate method ofobtaining the absorption and reduced scattering coef-

1 November 1994 / Vol. 33, No. 31 / APPLIED OPTICS 7397

ficients.10"14 This method permits the use of colli-mated or diffuse incident light to measure the totalreflectance and transmittance of the sample. Withthe addition of an on-axis collimated transmissionmeasurement, which determines the total attenuatedcoefficient, Pq = jLa + [i,, the anisotropy factor can alsobe derived.

In this paper we present an improved integratingsphere and collimated beam technique, which allowsaccurate and fast measurement of the absorption andscattering coefficients as well as the anisotropy factorfor bronchial tissue and tumor over a relatively widespectral range. This technique may also be used tomeasure optical properties of other turbid materials.

Materials: Sample Preparation

All samples used in our measurements were obtainedwithin 4 h after lung resection from eight subjects.The samples were rinsed briefly in saline to removeexcess blood, snap-frozen in liquid nitrogen, andstored at - 70 'C until the onset of the experiments.The bronchus consists of three layers: a thin epithe-lium lining on the inside, then a thicker submucosa ofdense layers of mucous glands and smooth muscle,and, on the bottom, horseshoe-shaped cartilage cres-cents. Each layer must be separated sequentiallyfrom the others so that its optical properties can bemeasured separately. The tumor samples were largeenough to be separated from the underlying mucosaand cartilage.

The thickness of normal epithelium is only approxi-mately 50-70 jLm. To separate the epithelium byusing a microtome, one must adjust the depth controlprecisely to avoid the inclusion of any submucosa inthe epithelium sample. The white-light and inducedfluorescence images of an unstained histology sectionof epithelium and submucosa are shown in Fig. 1.As we can see from the figure, the submucosa is richin fluorophores, whereas the epithelium emits verylittle fluorescence. This information was used torecognize epithelium and submucosa during samplepreparation. In our experiments, every section ofepithelium cut from each bronchial tissue sample waschecked for laser-induced autofluorescence to ensurethat no submucosa was included in the section. Themaximum epithelium sample thickness obtained, freeof submucosa, was 30 jim. The sections of submu-cosa, cartilage, and tumor were also cut by microtome.The thin sections were cut from the center of theparticular structures to ensure submucosa, cartilage,and tumor sample sections. The thickness of submu-cosa, cartilage, and tumor samples varied from 100 to500 ,um. Each sample piece was sandwiched by apair of microscope slides, and a small drop of phos-phate-buffered saline was added to prevent air bubblesfrom forming between the tissue and the slides.This assembly was mounted into a special holder, asshown in Fig. 2, and used to perform the integratingsphere and on-axis transmission measurements.This specially designed slide holder not only kept thesample uniform, both in thickness and extent, but

(a)

(b)

Fig. 1. Histology and laser-induced fluorescence of bronchialtissue: (a) white-light imaging, (b) fluorescence imaging of the

same section excited by the 435-nm line of a Hg lamp and filteredby a long-pass filter.

also minimized sample compression. We measured9 epithelium, 15 submucosa, 12 cartilage, and 10tumor samples.

Methods: Total Reflectance andTransmittance Measurement

The employment of an integrating sphere is a tradi-tional technique to obtain the total reflectance andtransmittance of a sample. The tissue is positionedat a port of the integrating sphere and illuminated bya collimated or diffused light source. The diffuse

Screw Sample Screw

Slides

L-V SpringsFig. 2. View of the A-A section of the tissue-sample holder.

7398 APPLIED OPTICS / Vol. 33, No. 31 / 1 November 1994

reflectance and transmittance are then measured.' 3

The light field in an integrating sphere with a portoccupied by a sample is no longer spherically symmet-ric. The collection efficiency of an integrating spherewith a port occupied by a sample is lower than that ofan integrating sphere in which the sample port isoccupied by a standard plate. The experimentalerror caused by this factor in the calculation of thecollection efficiency must be corrected for all integrat-ing sphere measurements.

Very recently, an accurate measurement of opticalproperties of turbid media that used two integratingspheres was reported by Pickering et al.,'0 who dem-onstrated how the experimental errors in the reflec-tance and transmittance caused by the lower collec-tion efficiency of the spheres with the port occupiedby the sample can be corrected. In the experimentalsetup of Pikhering and co-workers, a large and opti-cally uniform sample is required to cover the sampleport to prevent a substantial loss of light around theedges of the sample.'0"15 However, in most cases, itis difficult to get large and optically uniform tissue.For example, for bronchial tissue only small sectionsof an irregular shape can be obtained, which requiresthat the illumination be limited to the opticallyuniform areas. The two-sphere technique that usesa large area of diffuse illumination is therefore notsuitable for lung-tissue measurements.

The experimental arrangements used for the mea-surement of reflectance and transmittance are shownin Fig. 3. Collimated illumination was used. Fromthe setup in Fig. 3(a), the reflection signal of thesample, R8, and the signal from a standard plate, Ro,can be obtained. The transmission signal for thesample, T, can be obtained through the use of thesetup in Fig. 3(b). In the setup shown in Fig. 3(c),the collimated beam is incident upon the sphere wallclose to the sample port. The signal with a sample ora standard plate covering the port, C and C, respec-tively, can be obtained. These measurements allowone to calculate the difference in collection efficiencyof the sphere with a sample at the port and with astandard plate at the port, (C - C)/(C 0 - Cb); Cb isthe background signal for the setup in Fig. 3(a)

Sample Sample

7Baffle Baffle

to MA to OMA

(a) (b)

to OMA

(C)

Fig. 3. Integrating sphere measurements: (a) collimated beam isincident upon the tissue for the total reflectance measurements; (b)collimated beam illuminates the sample from outside the spherefor the total transmittance measurements; (c) light beam isincident upon the sphere wall for the collection efficiency measure-ments of the sphere. In all cases a baffle was placed between theincident spot of the light beam and the detector.

measured with an empty port (equivalent to 0%reflection). In this research, (C, - Cb)/(C0 - C5) wasfound in the range from 88 to 91%, which wasdependent on the size and thickness of tissue samples.The total reflectance and transmittance of the sampleare given by

Rs- CbTT.,R - b

Cs - Cb'

C, - Cb

Co - Cb'(1)

(2)

where Tb is the background signal when the illumina-tion beam is blocked for the set shown in Fig. 3(b),and (C, - C5)/(C. - Cb) has been used to correct thedifference in collection efficiency of the sphere with asample at the port and with a standard plate at theport in Eq. (1).

Our experimental setup is shown in Fig. 4. Thetissue sample was placed at the exit or entrance portof the integrating sphere (Oriel Model 70491). Thediameters of the integrating sphere and the sampleport were 20.3 and 2.5 cm, respectively. The lightsource was a collimated broadband light beam. Thebeam size could be adjusted from 1 to 3 mm continu-ously through the use of two collimation apertures.The beam size used depended on the size and opticaluniformity of the sample. The ratio of sample areato beam area was kept in the range from 10 to 15.The divergent angle of the beam was approximately10 mrad. The signal from the integrating spherewas conducted by a 1-mm-diameter optical fiber tothe entrance of the spectrometer. The full collectionangle of the fiber is approximately 25°. The signalwas analyzed by an optical multichannel analyzer(OMA) system (OMA III, EG&G Princeton AppliedResearch Corp.). A standard diffusion reflector (OrielModel 5735B) was used to calibrate the reflectancesignals from the tissue samples. The reflectance andtransmittance of all samples included the contribu-

OMA Detector

pectromete Controller Computer

Optical Fiber LighSource

- - - n Optical Fiber

| I Collimator

Integrating Sphere SystemFig. 4. Experimental setup for the measurements of total reflec-tance and transmittance.


tion of specular reflection caused by refractive indexmismatching between the slides and the tissue.

Measurement of Unscattered Transmittance

The total attenuation coefficient pl = la + PUs, can beobtained from the unscattered transmittance mea-surement. However, if the thickness of the sampleis greater than the mean-free path of photons insidethe tissue, the photons may be scattered more thanonce. The propagation of photons in thick tissue is amultiple-scattering process. The on-axis transmit-tance will include the unscattered photons, whichmay be used to find the total attenuation coefficient aswell as the scattered factor that is caused by multiple-scattering events. For a sample of 280-[im-thickhuman dermis, the intensity of the scattered compo-nent of the on-axis transmittance is almost the sameas the unscattered transmission.' Three differentmethods have been developed to reduce the scatteredcomponent: goniometry, the measurement of verythin samples, and the positioning of the detector faraway from the tissue. The first two methods requireoptically thin samples and neglect multiple scatter-ing; it is difficult to cut a thin and optically uniformtissue sample. We found the measurement accuracyhad been strongly influenced by the optical unifor-mity of the sample. For the thicker and more opti-cally uniform sample, the last method not only re-quires a well-collimated light source but also makes itdifficult to estimate the residual scattered componentin the on-axis transmission. In our experiments, wedeveloped a polarized illumination and detection tech-nique to subtract the scattered component from theon-axis transmission.

From earlier studies'6' 8 it is known that photonswill completely lose their coherence and partly losetheir polarization when scattered. The length of thepath over which a photon becomes depolarized de-pends not only on whether initially it is linearly orcircularly polarized but also on the scattering coeffi-cient and the scattering anisotropy of the particleswith which it interacts. A large number of scatter-ing events are required to randomize the polarizationof a circularly polarized photon, compared with thenumber required to randomize the polarization of alinearly polarized photon. If the incident light beamis a linearly polarized source, the transmission shouldinclude a polarized fraction and an unpolarized scat-tered component. The transmission components forlight perpendicular and parallel to the polarizationaxis of the incident beam are

Ti 1= Tunscatter + Tscattered,

aL = Tscattered/TI,

(3)

(4)

where To and TI, are the transmissions on the axisperpendicular and parallel to the incident polariza-tion axis, respectively. Here Tunscatter is the totalunscattered transmittance, Tscattered is the scatteredcomponent on the axis parallel to the incident polar-ization axis, and a is the polarizability of the scattered

photon on the axis, which may be extrapolated fromthe polarizability of near-axis scatter-ing light. Theunscattered component can be obtained from themeasurement of T,, a, and TI1, and Tunscatter = T 1 -

aT,.Our experimental setup is shown in Fig. 5. Two

polarizers (Melles-Griot Model 03 FPG 007) wereplaced in front of a collimated light source and opticalfiber coupler, respectively. The tissue sample, colli-mation apertures, and optical fiber coupler were on agonimetric apparatus, made up of a rotation stage(Melles-Griot Model 07 TRT 002) and a small opticaltable rail that was rigidly connected with the stage.The sample was placed in the center of the rotationstage. The collimation apertures, fiber coupler, andpolarizer in front of the coupler were fixed on anoptical table rail. The collimated source was thesame as that used in the integrating sphere measure-ments. The light beam was incident perpendicularto the sample's surface. The size of the beam and allapertures were adjusted to 1 mm. The distancebetween the sample and detector was 30 cm. Thesolid angle of the detector was approximately 1.1 x10-5 sr. The transmission signal collected by theoptical fiber coupler was analyzed by the OMA system.All samples of the epithelium, submucosa, cartilage,and tumor were again sandwiched by a sample holderas shown in Fig. 2.

To estimate a, the polarizability of scattered pho-tons on axis, we measured the polarizability of near-axis scattering light. The detector was positioned atangles from -3° to 3° relative to the forward on-axisdirection of the incident light beam in increments of0.5°. The light signals parallel and perpendicular tothe polarization axis of incident light, S 1 and S,, wererecorded. The polarizability at different scatteringangles was calculated by S, /S,, which was found tovary by a small amount at the near-axis scatteringangle in the 0.5-3° range. Then a values of allsubmucosa, cartilage, and tumor samples were ex-trapolated from the polarizability of near-axis scatter-ing light through the use of a quadratic fittingfunction. During the measurement of near-axis po-larizability, the effects of refractive index mismatch-ing at the tissue-slide-air boundary were corrected.

In the measurements of transmission and scatter-ing of all tissues, a set of neutral-density filters (Oriel

to OMA

Polarizer

Optical FiberCoupler

to White-Light Source

Polarizer

Collimator

Sample

Fig. 5. Experimental setup for the measurements of the unscat-tered transmittance of the tissue; the optical table rail is not

shown.


II

Models 50800-50850) with optical densities from 0.3to 5 were used to calibrate the absolute transmittance.The total attenuation coefficient, jit = a + .,L, wasobtained by

R = - ln(TI - aT)/d,where d is the sample thickness.

1.0

0.8 F

0.6 .

(5) cC

Results and Discussion

The IAD method of determination of the opticalproperties from the three measurements of reflec-tance, transmittance, and unscattered transmittanceas well as sample thickness has been discussed indetail in Refs. 10 and 14. The typical reflectance,transmittance, and unscattered transmittance of epi-thelium, submocosa, and cartilage as well as tumorare shown in Figs. 6(a)-6(d). In the IAD solution forthe radiative transfer equation, the refractive indexof the sample was assumed to be 1.37, and the indexof air and slides was 1.0 and 1.54, respectively.

It has been found that the IAD solution becameunstable if the sample was very thin. One couldamplify the error in the measurements of thin samplesdramatically by using the IAD method to the point ofcausing solution divergence. For the epithelium mea-surements, the thickness of the sample was only 30jim. We found that the absorption coefficient for theIAD solution was extremely sensitive to the totaltransmittance. The 1.5% error associated with thereflectance and transmittance measurements couldcause over 100% error for the calculation of theabsorption coefficient. However, the reduced scatter-ing coefficient of the IAD solution was not as sensitiveto the errors in the total reflectance and transmittance.The error associated with the reduced scatteringcoefficient was below 15%. This also suggests thatthe scattering process dominates in the epithelium,and ALta is small compared with , and Wt,. Theanisotropic factor, g, may be calculated from( - i.L/"Lt).

Figures 7(a)-7(c) show the absorption coefficient,the scattering coefficient, and the anisotropic factorfor the epithelium as a function of wavelength. Overthe 400-450-nm range, the IAD solution does notconverge. The optical coefficients were extrapolatedfrom the values found in the 450-550-nm range.The mean optical properties of the submucosa, carti-lage, and tumor are presented in Figs. 7(d)-7(l). Themean and standard deviations of optical propertyspectra for each group of samples were calculatedfrom the average of three measurements on eachtissue sample.

The absorption coefficient spectrum of the submu-cosa [Fig. 7(e)] shows double peaks in the 540-590 nmrange. The reason for this increased absorption isthat the submucosa is a blood-rich tissue, for whichthe absorption spectrum is a superposition of thegently sloped absorption spectrum of the tissue andthe absorption caused by the residual blood that hadnot been rinsed out during our sample preparation.The relatively larger error of the absorption in this

0.4

0.2

0.0 40

Transmittance

Reflectance

0 460 520 580

Wavelength (nm)(a)

1 .0

00.8 a

0

E0.6 c

0.4 2e

0.2 cD_

I . I 0.0640 700

0

0IDC

a0

a)C00

Wavelength (nm)(b)

1.0

0.8

0

0

0.6

0.4

0.2

0.0 _400

1.0

0.8

0-

a,

0.6

0.4

0.2

0.0 _400

460 520 580

Wavelength (nm)(c)

460 520 580Wavelength (nm)

(d)

1 .OE-3

28.OE-4 C

E6.OE-4 2

4.0OE-40

02.OE-4 c

M

' 0.0EO640 700

1.OE-3

8.OE-4 c

- ~~E- 6.OE-4 c

sctance - 4.0E-4 c

2.OE-4 0

640 700

Fig. 6. Typical total reflectance, total transmittance, and unscat-tered transmittance (dashed curve) of bronchial tissue: (a) epithe-lium, (b) submucosa, (c) cartilage, (d) tumor.


. . . . . . ., .

400 450 500 550 600 650 700

Wavelength (nm)(a)

400 450 500 550 600 650 700

Wavelength (nm)

(b)

400 450 500 550 600 650 700

4

3

750

cm1

.20

co

0

.CCUc)'

0a

750

1.00

0.98

0.96

0.94

0.92

0.90

0.88

0.86

0.84

0.82

0.8035

Wavelength (nm)

(e). I - . . 1 . . I . I I

a

Wavelength (nm)(f)

400

300 -

E200 -

100

035'

750 0

Wavelength (nm)(C)

.. . . .. . . . . . . . . . .~~~~~~~~~~~~~~~~

400 450 500 550 600 650 700

Wavelength (nm)(d)

750

101

4

S 3

:1 2

Wavelength (nm)

(h)

Fig. 7. Optical properties of epithelium, submucosa, cartilage, and tumor versus wavelength over the 400-700 nm range: (a) scattering

coefficient, Lu, of the epithelium (dashed curve indicates the extrapolated values), (b) absorption coefficient, jla, of the epithelium (solid curve

was obtained with a quadratic fitting function), (c) anisotropic factor, g, of the epithelium, (d) Lu of the submucosa, (e) Bu, of the submucosa,

(f) g of the submucosa, (g) ps, of the cartilage, (h) Au of the cartilage, (i) g of the cartilage, (j) H, of the tumor, (k) An0 of the tumor, (1) g of the

tumor.

500

400

E-a

300

200

4

3

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n350

400 450 500 550 600 650 700

0)

CU

CL

-, * - ' ' '

..... ........ A

0.99

0.97

0.95

0.93

0.91

0.89

0.87

0.8535

750

0

JlihIJIiIi±IIIIIIIITTJTJHJT

400

300

E 200

400 450 500 550 600 650 700

Wavelength (nm)

(9)

750

100

n L

350

7 . 0 4 6 6

350 400 450 500 550 600 650 700 750

. ~ ~ ~ ~ .. . . . . .

| | l | w l l

. . I . . . . . I . . . I .

. \ 'I11

, II�' -1 "

I I . . . . . I . I . I . I . . . . . .

. I . . . . . . .. . . . . . . . . . . . .

. I . I . I . I . I . .

100

0350

........

. I

H

50 400 450 500 550 600Wavelength (nm)

(i)

- ~ ~ ~ -- - - -.

0 400 450 500 550 600

Wavelength (nm)

(i)

400 450 500 550 600

Wavelength (nm)(k)

range may be explained by the slightly differentvolumes of excess blood in each submucosa sample.There is no blood present in the cartilage, andtherefore its absorption characteristic as a function ofwavelength is smooth. The absorption spectrum ofthe tumor also shows a relatively weak blood absorp-tion because there is less blood in poorly vascularizedor necrotic tumor tissue. The behavior of the scatter-ing coefficient and the g factor for the four tissuetypes are similar to the behavior of the total attenua-tion coefficient. These vary slowly, because [s =

650 700 750 i A - Ia, g = Ls'/ 1 - and la is much smallercompared with At

To our knowledge, there has been little informationpublished on the optical properties of bronchial andtumoral tissue. In Table 1, optical properties of lungtissue are compared with the values published byMarchesini et al. 19 In their research, an integratingsphere was used to obtain the absorbance of tissue,the unscattered transmittance was measured by gonio-photometry, and the absorption coefficient was de-rived from Beer's law. The optical properties re-ported by Marchesini et al. are significantly higherthan those found in this study. The absorptioncoefficients measured by Marchesini et al. were ob-tained from sample absorbance by using Beer's law;in this way, the scattering of tissue can increase the

550 700 750 effective path lengths of photons inside the tissue.Thus the absorption coefficient will be overestimated.

The optical properties affected by the errors in theexperimental data have been discussed in Refs. 20and 21. For g = 0.875 and albedo a = 0.9, a =Is4L/(ls + a), a 5-10% change in total reflection canchange the calculated total attenuation coefficient bya factor of 2.21 This suggests that a small error inthe measurements of reflectance can produce a factorof 2 change in the albedo, and the scattering coeffi-cient becomes sensitive to the errors in the measure-ment of reflection.20

In our experiments, we found that the collectionefficiency of the integrating sphere with a tissuesample covering the port decreased to 90% of that of

650 700 750 the integrating sphere in which the port is covered bya standard plate because of the loss of the lightleakage and the low reflection of the tissue. This

400 450 500 550 600 650 700 750

Wavelength (nm)

(I)

Fig. 7 continued.

Table 1. Published Optical Properties at Selected WavelengthsCompared with the Values in this Study

X P-a PAsParameter (nm) (cm-') (cm' ) g

Lung Tissuea 633 8.1 ± 2.8 324 ± 46 0.75Epithelium 633 1.1 ± 0.5 204 ± 32 0.95 ± 0.01Submucosa 633 1.8 ± 0.2 207 ± 13 0.94 ± 0.01Cartilage 633 1.0 ± 0.1 246 ± 16 0.95 ± 0.01Tumor 633 1.2 ± 0.2 240 ± 15 0.95 ± 0.01Lung Tissuea 515 25.5 ± 3.0 356 ± 39Epithelium 515 1.6 ± 0.7 273 ± 42 0.94 ± 0.01Submucosa 515 3.7 ± 0.5 236 ± 18 0.92 ± 0.01Cartilage 515 2.1 ± 0.2 270 ± 18 0.93 ± 0.01Tumor 515 2.6 ± 0.3 265 ± 17 0.94 ± 0.01

aRef. 19.


11

0

.CU

00r

1.00

0.98

0.96

0.94

0.92

0.90

0.88

0.86

0.84

0.82

u.eu

400 -

300 -

ILl

200

100 -

035'

2

1 01

76

° 4

3

10;

350

11

0

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1.00

0.98

0.96

0.94

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0.84

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0.8035'

. . . . . . . . I

. . I . . . . . .

. . I . . . . . .

. . . . . . . . . . .

. . . . . . . . . . . .

iO

. . . . . . . I . . . . . I .

would cause an approximately 10% error in thereflectance measurements. Table 2 shows coeffi-cients ig,, and [L' determined by IAD from a set ofcorrected and uncorrected total diffuse reflectancemeasurements of submucosa at selected wavelengthsin the 450-650 nm range. The averaged effect of thecorrected versus the uncorrected reflectance measure-ments on jia and jil' reflectance are 18% and 11%,respectively. The errors increased as the albedoincreased. In this study the collection efficiency ofthe sphere with sample was corrected in Eqs. (1) and(2). We checked our correction technique by measur-ing the reflection of a slide. The reflection of theslide is specified by Fresnel reflectance, and it amountsto 8.8% for the conditions of this experiment. Themeasured reflectance found through the use of Eq. (1)is 8.7%, and for the uncorrected equation it is 7.9%.

An optical fiber, a nonisotropic detector, was usedto collect the signal from the integrating sphere.The influence of different detectors on signal-collec-tion efficiency was discussed in Ref. 22. For colli-mated illumination, the power detected is dependenton the point of incidence of the light beam. In ourmeasurement, the total reflectance, total transmit-tance, and reference signals in Eqs. (1) and (2) wereobtained when the point of incidence light was on orvery near the sample port. Thus the detection signalby optical fiber should be equivalent to isotropicdetection.

The thickness of the sample is another importantfactor strongly influencing the accuracy of the opticalproperty measurements. There is considerable vari-ability in the measurements of the absorption as afunction of sample thickness.20 We believe that therelatively large errors in the optical properties for theepithelium were due to the optical nonhomogeneity ofthe thin epithelium samples. Thus, optically thicktissue is recommended for the measurement becauseit is not only more optically uniform but also pre-serves the original tissue structure. The maximumthickness of tissue is limited by the detectability ofthe total diffusion transmittance and on-axis transmit-tance. However, for optically thick tissue, it is impos-sible for the commonly used on-axis transmissionmeasurement to estimate the residual scattered com-ponent of the on-axis transmittance. In this studythe polarization detection technique was used toextract the unscattered transmittance from the on-axis transmittance. We checked this technique by

Table 2. Coefficients C and js,' Determined by lAD from the Correctedand Uncorrected Total Diffusion Reflectance

X (nm)

Coefficient 450 500 550 600 650

Placorrected 7.48 4.36 4.32 2.08 1.923

uncorrected 8.35 5.03 4.78 2.57 2.51

P-s

corrected 22.1 18.1 16.5 13.9 12.4

uncorrected 19.7 16.3 14.7 12.4 11.1

measuring the scattering coefficient of 1-jLm-diam-eter sphere (Duke Scientific) suspensions in water.

The macroscopic scattering coefficient of suspen-sions of microspheres in water is linearly propor-tional to the concentration of spheres. One cancontrol the scattering coefficient of the suspensionsby changing the concentration of microspheres. Theanisotropic factor of a 1-jm-diameter sphere is 0.919at a 630-nm wavelength,'8 which is close to the gfactor of human tissue. The suspensions of micro-spheres in distilled water were put into a 2.4-mm-thick container. The entrance and exit surfaces ofthe container were made of optical-quality glass.Other surfaces were constructed from black rubber,which reduced internal reflections. Figure 8 showsthe on-axis transmittance and unscattered transmit-tance at 630 nm measured by the polarized detectiontechnique as a function of the scattering coefficient ofthe suspensions. On-axis transmission measure-ment worked well in the low-scattering range becausethe residual scattering component was much smallerthan the unscattered transmittance. However, thedifference between on-axis transmittance and unscat-tered transmittance increased rapidly in the high-scattering range because on-axis transmission in-cluded a residual scattering component that could notbe neglected from the high-scattering range. If theresidual scattering component is not extracted fromon-axis transmittance, the measured scattering coef-ficient will be significantly underestimated. By us-ing the polarized detection method, we found that theaccuracy of the measured unscattered transmittancewas much higher than that obtained by on-axistransmission measurements in the high-scatteringcoefficient range. As seen in Fig. 8, our polarizeddetection technique could extract the unscatteredtransmittance from on-axis transmittance even whenthe residual scattering component was ten timeshigher than the unscattered transmittance.

In this experiment the unscattered transmittance

1 00

10-1

0

E0orc-

1 0-2

1 0-3

1 0-4

1 0-5

1 0-8

10-70 10 20 30 40 50 60 70

Scattering coefficIent (cm-i)

Fig. 8. Transmittance of suspensions of 1-P-m-diameter spheresin water: *, on-axis transmittance; *, transmittance measuredby the polarized detection method. The solid curve is an exponen-

tial function fitted to the on-axis transmittance measured over the

range of scattering coefficients from 0 to 16 cln- 1.


S~~~~

0

I L_ I - I I~~~

was detectable through the use of the polarizeddetection method when the scattering coefficient wasless than 70 cm-1 . During our tissue measurement,we found that the unscattered transmittance couldnot be detected when the sample thickness was over400 lm. All optical properties shown in Fig. 7 werecalculated from the data measured from samples withthicknesses ranging from 100 to 300 jim.

Conclusion

Optical properties of bronchial tissue and tumor weremeasured in the range from 400 to 700 nm. Thevalues of the optical properties were found to besensitive to errors in the total reflectance measure-ments, especially for high-albedo samples. By cor-recting for the effects of the sample on the integratingsphere measurements, we found that the accuracy ofthe optical property measurements increased signifi-cantly. The polarization detection technique de-scribed in this study offers a means to obtain theunscattered transmittance of optically thick tissueand accurately specify the total attenuation coefficient.A thick sample is recommended for the measurementto reduce the error produced by the optical nonhomo-geneity of thin samples, in which the original struc-ture of the tissue may have been destroyed.

This research was supported through grants pro-vided by the Medical Research Council of Canada (MA91348) and the British Columbia Lung Association.We are also grateful for financial support from XillixTechnologies Corporation. We thank S. Prahl forproviding the IAD code; R. Miller, Department ofPathology and Thoracic Surgery, for supplying thebronchial specimens used in this study; and B.Thomas and A. Choi for their assistance with tissuepreparation.

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optical properties of normal and carcinomatous bronchial tissue

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