Research Papers: Sensing

Enhancement factor in low-coherence enhanced backscattering and its applications for characterizing experimental skin carcinogenesis

[+] Author Affiliations
Jingjing Liu, Zhengbin Xu, Qinghai Song

Purdue University, Weldon School of Biomedical Engineering, West Lafayette, Indiana 47907

Raymond L. Konger

Indiana University School of Medicine, Department of Pathology and Laboratory Medicine, Indianapolis, Indiana 46202

Young L. Kim

Purdue University, Weldon School of Biomedical Engineering, West Lafayette, Indiana 47907

J. Biomed. Opt. 15(3), 037011 (June 09, 2010). doi:10.1117/1.3443795
History: Received November 11, 2009; Revised April 12, 2010; Accepted April 20, 2010; Published June 09, 2010; Online June 09, 2010
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* Address all correspondence to Young L. Kim, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907. Tel:765-496-2445; Fax:765-496-1496; E-mail: youngkim@purdue.edu

We experimentally study potential mechanisms by which the enhancement factor in low-coherence enhanced backscattering (LEBS) can probe subtle variations in radial intensity distribution in weakly scattering media. We use enhanced backscattering of light by implementing either (1) low spatial coherence illumination or (2) multiple spatially independent detections using a microlens array under spatially coherent illumination. We show that the enhancement factor in these configurations is a measure of the integrated intensity within the localized coherence or detection area, which can exhibit strong dependence on small perturbations in scattering properties. To further evaluate the utility of the LEBS enhancement factor, we use a well-established animal model of cutaneous two-stage chemical carcinogenesis. In this pilot study, we demonstrate that the LEBS enhancement factor can be substantially altered at a stage of preneoplasia. Our animal result supports the idea that early carcinogenesis can cause subtle alterations in the scattering properties that can be captured by the LEBS enhancement factor. Thus, the LEBS enhancement factor has the potential as an easily measurable biomarker in skin carcinogenesis.

Figures in this Article

Light propagation in biological tissue and light-tissue interaction are commonly studied in relatively large volumes, for example, in a few cubic millimeters. In diffusion approximations for tissue optics, the radial distance distribution p(r) with r=radial distance are commonly described by a function of ls*. In this respect, there have been numerous methods to measure macroscopic optical properties such as the transport mean free path of light ls* in biological media to obtain information about physiological conditions and disease status.15 Such measurements are ideal to study the profiles of p(r) when rls* as intended to capture the behavior of diffusive light in biological media. On the other hand, it is challenging to obtain information about p(r) when rls*, in part because it is extremely difficult to generate a pencil-like illumination of 100to300μm in diameter due to the diffraction. There can be instances in which a subtle alteration in p(rls*) may not be sensitively captured by the diffusion-approximation-based methods. To easily obtain p(rls*) for tissue characterizations, we can use enhanced backscattering of light (EBS, otherwise known as coherent backscattering of light, CBS). EBS is a robust manifestation of self-interference effects in elastic light scattering, which gives rise to an enhanced scattered intensity in the backward direction.68 For a plane wave incident on a semi-infinite random medium, every wave scattered from the medium in the backward direction has its counterpart time-reversed wave traveling along the same path in the opposite direction. These waves have consistent phase differences at the exit points and thus interfere constructively with each other. Consequently, the angular profile of the EBS peak can be considered a Fourier transform of p(r). The width of the EBS peak is inversely proportional to the product of the wave number κ and the transport mean free path of light in the medium ls*. In principle, the EBS peak amplitude can be as high as 2, compared with the incoherent baseline. In particular, to detect a variation in p(r) when rls*, we can use the enhancement factor [i.e., the ratio of the exact backscattering intensity I(θ=0deg) to the incoherent baseline intensity] in EBS measurements under low-spatial-coherence illumination (hereafter it is referred to as low-coherence enhanced backscattering, LEBS). The angular profile of LEBS can be expressed as ILEBS(θ)0C(r)rp(r)exp(i2πrθλ)dr, where C(r)=|2J1(rLsc)(rLsc)|, J1 is the first-order Bessel function, Lsc is spatial coherence length, and θ is the backscattering angle.911 Thus, the LEBS enhancement factor 1+0C(r)rp(r)dr0rp(r)dr. In other words, the LEBS enhancement factor is a measure of the intensity within the spatial coherence area, which is also normalized by the incoherent background intensity. Any changes in the LEBS enhancement factor are directly related to changes in p(r<Lsc). In our previous studies,1116 low-spatial-coherence illumination was implemented in EBS to isolate low-order scattering by dephasing the conjugated time-reversed paths outside its spatially coherent area when Lscls*. In this case, the coherent area (or the transverse modes) can be as small as Lsc2=502to1002μm2, while ls* is of the order of a few millimeters in biological tissue. In general, a change in p(r) could be originated from variations in the macroscopic optical properties such as ls*. On the other hand, when rls*, there can be a case in which a subtle alteration in p(r) may not be sensitively captured by the macroscopic optical property. Figure 1 illustrates the instance in which the LEBS enhancement factor measurements can be distinct from conventional EBS measurements. In conventional EBS, the probing volume of the time-reversed interference waves is similar with those of the incoherent waves, while low-spatial-coherence illumination generates a discrepancy between the coherent and incoherent volumes. The LEBS enhancement factor provides averaged information of the local optical properties that are determined by the multiple individual coherence volumes. This averaging effect partially preserves the local optical properties as compared with the averaging over the entire area. Collectively, the LEBS enhancement factor can be sensitive to subtle alterations in light propagation, in particular, when rls*.

Grahic Jump LocationF1 :

Schematic illustration of the instance in which the LEBS enhancement factor can be distinct from the EBS enhancement factor under spatially coherent illumination. The two media [sketched in (a) and (d)] consist of scatterers of different relative refractive indices nr, different diameters Dp, and different numbers of particles (per milliliter), while the transport mean free path ls* and the anisotropic factor g are identical. In this case, (b) and (e) show the identical angular profiles with the enhancement factor of 2 because of the same ls*. On the other hand, the LEBS enhancement factors in the two media can be different, because the relative small coherent volumes determined by low-spatial-coherence illumination partially preserves the local optical properties as compared with the conventional measurement in the large volume.

In this paper, to study the LEBS enhancement factor [i.e., p(r<Lsc)] in weakly scattering media including early cancerous tissue, we implement (1) low-spatial-coherence illumination using a finite size of an illumination source and (2) multiple spatially independent detections using a microlens array under spatially coherent illumination. We investigate the enhancement factor in these configurations from various optical properties of scattering media. In particular, a microlens array in the detection arm under spatially coherent illumination enables us to experimentally study the angular distribution of coherent light propagation within each independent localized area. Each microlens can serve as a spatial filter, which provides a unique configuration to study scattering properties of the media in a localized area as similarly as low-spatial-coherence illumination. Finally, we report a pilot animal study of nonmelanoma skin cancer using two-stage carcinogen treatments to investigate changes in the LEBS enhancement factor during early skin carcinogenesis.

Experimental Setups

Our first experimental setup for LEBS measurements is described in Fig. 2. In brief, a beam of broadband cw light was linearly polarized and delivered onto a sample with the illumination diameter of 4mm. By changing aperture size of A1 in the 4-f lens system, we obtained two values of Lsc of the incident light (Lsc=115 and 200μm). An interference filter was placed in the delivery part to select the central wavelength of 628nm with the spectral width of 40nm. The value of Lsc was confirmed by the Young’s double-pinhole interference experiments.17 The light backscattered by the sample was collected by a sequence of a lens, a linear analyzer parallel to the incident polarization direction, and a CCD camera. We analyzed angular intensity profiles with the polarization direction parallel to that of the incident light. As shown in Fig. 2, we further demonstrated that the multiple localized volume detections played a key role in LEBS measurements as follows. We placed a microlens array between the 4-f lens system and the relay lens system using spatially coherent illumination from a He-Ne laser. The microlens array (pitch, 250μm, and area, 10×10mm) was placed exactly on the imaging plane of the sample so that each microlens at different spatial locations additionally served as a spatial filter for the time-reversed interference signals. The relay lens system was used to map the focal plane of the microlens array onto the CCD camera. More importantly, this configuration can be used to study angular dependency of light scattered from the sample over different spatial locations. Thus, our two complementary experimental setups enabled us to investigate EBS formation in the regime of rls* by altering either the illumination property or the detection configuration. For the animal studies, as shown in Fig. 2, the lens in the detection arm directly projected the angular distribution of the backscattered light onto the slit of the spectrograph, which dispersed the light according to its wavelength in the direction perpendicular to the slit of the spectrograph. Thus, angular distribution of backscattering light in a certain wavelength range can be selected. In particular, we can avoid the spectral range that is affected by the hemoglobin absorption band. For the pilot animal study, we used the values of Lsc=120μm to study carcinogenesis in the superficial skin.

Grahic Jump LocationF2 :

Complementary experimental setups using (a) EBS measurements under low-spatial-coherence illumination (i.e., LEBS), (b) multiple spatially independent detections using a microlens array, and (c) LEBS spectroscopy instrument. (a) The aperture A1 determines the spatial coherence length of illumination. A single lens L projects the angular distribution of the backscattering light onto the CCD camera. (b) The microlens array in the detection arm offers multiple mutually independent spatial filters under spatially coherent illumination. (c) The lens in the detection arm projects the angular distribution of the backscattered light onto the slit of the spectrograph, which disperses the light according to its wavelength in the direction perpendicular to the slit. The CCD records an intensity matrix as a function of the wavelength and the backscattering angle. P1 and P2 are polarizers. A1 and A2 are apertures. F is an interference filter.

Phantom Preparations

We studied that the LEBS enhancement factor can be sensitive to subtle alterations in p(r<Lscls*) resulted from changes in either the macroscopic scattering properties such as ls*, or the internal structures such as the relative refractive indices and the scatterer sizes. We mixed microspheres with different amounts of the solvents, and varied the relative refractive index nr (=npnm, where np and nm are the refractive indices of the particle and the medium, respectively) and the particle size Dp. In particular, to study the effect of a minute perturbation by nr and Dp alone, we must keep the macroscopic scattering properties identical. In this case, we used 0.87-μm polystyrene microspheres (np=1.58, Ref. 18) and 0.78-μm silica microspheres (np=1.46, Ref. 19) as the scatterers and distilled water or glucose solution as the media. Table 1 illustrates representative sample preparations when ls*=400μm and g=0.91 at λ=628nm. To accurately measure nm of the mixed medium of glucose and water, we obtained elastic backscattering spectral data ranging from 450to650nm from the glucose suspension containing polystyrene microspheres of Dp=5.43μm. We fitted the experimental spectra with Mie theory simulations using the known particle diameter Dp and the known np of the large particles. Because the ripple spectral pattern from the glucose suspension of the large particles was highly dependent on nm, we could accurately estimate nm when the correlation coefficient between the simulation and the experimental spectra was the maximum. For the glucose suspension with the concentration of 3.11molL, we obtained its nm=1.38 at 628nm at room temperature. Using this procedure, we also confirmed nm=1.33 of water at room temperature.20 In the main study, we varied ls* from 32μmto1mm and fixed g of 0.91 to mimic most biological tissue.21 The macroscopic scattering properties were calculated using Mie theory.22 The dimension of the samples was approximately π×25mm2×13mm.

Table Grahic Jump Location
Representative sample preparations of the scattering media of ls*=400μm and g=0.91.

Since Lsc is an additional length scale in LEBS, we first experimentally confirmed that the ratio of ls* to Lsc can serve as a unitless length scale, which was also used in previous theoretical studies.910 Note that Lscls* is related to the ratio of the interference portion contributed by the coherent volume to the incoherent baseline. We tested that the unitless length scale Lscls* can determine the enhancement factor of LEBS. In this experiment, we prepared two different scattering media of ls*=400μm and ls*=700μm and measured the EBS angular profiles under Lsc=115μm and Lsc=200μm, respectively. Figure 3 shows the angular profiles from the two typical scattering media when Lscls*0.29. The enhancement factors of the two cases were statistically the same with pvalue=0.44, supporting the utility of Lscls*. Using the unitless parameter, we further studied the LEBS enhancement factors in the range of Lscls* from 0.2 to 6.25 using water suspension of polystyrene microsphere (nr=1.19 and Dp=0.87μm), as shown in Fig. 4. When Lscls*1, the enhancement factor was high and then decreased rapidly as Lscls* decreased. Even in the case of Lscls*1, there were significant dephasing effects manifested as the reduction in the enhancement factor, indicating that long scattering paths can be substantially destroyed in this regime. Our experimental results are also in good agreement with the theoretical studies.910

Grahic Jump LocationF3 :

Angular profiles obtained from the water suspension of polystyrene microspheres of ls*=400μm and ls*=700μm under two different values of Lsc=115μm and Lsc=200μm. Because ls*Lsc3.5 in the both cases, the enhancement factors are statistically the same.

Grahic Jump LocationF4 :

(a) Enhancement factor as a function of the unitless length scale Lscls* measured from the water suspension of polystyrene microspheres (nr=1.19) and (b) enhancement factor over the unitless length scale Lscls*<1 for three different nr and Dp. The LEBS enhancement factor depends on subtle perturbations of ls*, nr, or Dp.

To explore the unique instance in which a subtle alteration in p(r) may not be sensitively captured by the macroscopic scattering properties, we investigated the effect of nr and Dp on the enhancement factor when ls<Lsc<ls*. The regime of ls<Lsc<ls* covers most biological cases where ls is generally less than 100μm and ls* is greater than 1mm. Figure 4 shows that under the same macroscopic optical properties, the higher nr gives rise to a higher enhancement factor under low-spatial-coherence illumination. Indeed, when Lsc<ls*, the enhancement factor can be sensitive to subtle changes in nr and Dp. As shown in Fig. 4, the gradual increase in the enhancement factor over nr at a given Lscls* represents the increase in the local scattering power, which cannot be assessed using conventional EBS measurements. This is because the LEBS averages the multiple measurements of the local optical properties when rls*. This effect cannot be manifested if the optical properties are averaged in the entire area (e.g., Lscls*). From the perspective of conventional EBS, when the macroscopic optical properties of the scattering medium are kept the same, the EBS profile should remain the same with the enhancement factor of 2. Overall, the LEBS enhancement factor is directly related to a change in p(r<Lscls*), which can be originated from variations either in the macroscopic optical properties (e.g., ls*) or the sample structure details (e.g., relative refractive indices and sizes of scatterers). This characteristic of LEBS over a large range of Lscls* further supports the utility of the LEBS enhancement factor for tissue characterizations.

We also used the microlens array under spatially coherent illumination, as shown in Fig. 2. The microlens can serve as a spatial filter so that time-conjugate waves outside its finite aperture cannot interfere with each other. Similarly to EBS measurements under low-spatial-coherence illumination, Fig. 5 shows that the enhancement factor of each EBS cone is significantly reduced, because each microlens determines the coherent volume. The medium in Fig. 5 was a uniform suspension of polystyrene microspheres. The nonuniform peak profiles were due to the inconsistent focal lengths of each microlens and imaging aberrations. Overall, this configuration enabled us to calculate a matrix of the enhanced factors. We prepared two different media of different nr and Dp with the same macroscopic scattering properties of ls*=370μm and g=0.92 at 543nm. The values of ls* of both samples were confirmed to be the same by the identical angular profiles using the conventional EBS measurements. The enhancement factor was calculated by averaging the intensity from all the azimuthal angles. Figure 5 illustrates typical multiple EBS cones formed by each aperture of the microlens array when Lsc was much greater than the pitch size. We compared the two enhancement factor matrices pairwise, as shown in Fig. 6. Using paired t tests, the two enhancement factor matrices were statistically different with pvalue=4.64×105, although the macroscopic scattering properties were identical. This result was in good agreement with the previous LEBS enhancement factor measurements under low-spatial-coherence illumination. We note that the overall enhancement factors using the microlens array were lower than that of the single LEBS reading, probably due to low angular resolution through the series of the lenses. This result from the second experimental setup [Fig. 2] shows that each EBS peak in the matrix is mainly determined by the localized traveling waves, supporting the previous result that a single LEBS reading (i.e., the averaged value of many unit EBS cones) can detect the subtle changes in p(rls*). In addition, this configuration helps us to further understand the working principles of LEBS.

Grahic Jump LocationF5 :

Typical 3×3 angular profiles measured under spatially coherent illumination (i.e., He–Ne laser) using the microlens array that allows multiple spatially independent detections. The angular range of each EBS cone is from 3to3deg. The EBS cones are normalized by the intensity at a large scattering angle. The pitch of each microlens is 250μm.

Grahic Jump LocationF6 :

Enhancement factor matrix measured using the microlens array under spatially coherent illumination (i.e., Lscls*). Although the two media have the identical macroscopic scattering property (ls*=370μm and g=0.91 at 543nm), the multiple spatially independent detections reveal the influence of nr and Dp on the enhancement factor matrix.

Pilot Animal Study Using Cutaneous Two-Stage Chemical Carcinogenesis of Skin Cancer

To evaluate the utility of the LEBS enhancement factor for characterizing early carcinogenic alterations, we conducted a pilot animal study using an established animal model of nonmelanoma skin cancer (NMSC). Our animal study was approved by the Purdue Animal Care and Use Committee. Multistep models of carcinogenesis have been intensively studied23 in the mouse model of NMSC and the skin is the ideal organ to study carcinogenic alterations in vivo over time. We employed 7,12-dimethylbenz[a]anthracene (DMBA) + phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated mice as a model system for NMSC (Ref. 24) In our DMBA/TPA-treated mice, cancer initiation was accomplished by a single dose of DMBA on the dorsal skin, and cancer promotion was triggered by repeated applications of TPA, producing multiple papillomas. Because of strong similarities with human skin carcinogenesis, this highly reproducible animal model has been widely used in basic and clinical cancer research.23 Indeed, this is among the most intensively studied murine models of epithelial cancers and several ongoing human clinical trials are based on results generated by this animal model. In addition, in the previous LEBS studies,14,25 different animals or patients harboring different carcinogenesis stages were used without monitoring carcinogenesis over time. Because of the easy accessibility to the skin, our animal model enabled us to monitor each animal through several stages of carcinogenesis without sacrificing them at different time points, and to analyze the data specifically from the animals eventually harboring invasive tumors.

We used eight female FVB/n mice for this pilot study (three control mice and five treated mice). This strain is known to be particularly sensitive to two-stage chemical skin carcinogenesis.26 We had four mice for the DMBA/TPA-treated group and three mice for the age-matched control group. One mouse among the treated mice was used for histologic confirmation at 10weeks after the carcinogenesis initiation. At 6to7weeks of age, we shaved the dorsal skin of the mice using an electric hair shaver and completely removed hairs with a depilatory cream. One week after hair removal, we started the topical carcinogen administration as follows. The experimental group of the mice was treated with a single topical drop of 0.2ml acetone containing 25μg DMBA to the shaved dorsal skin. After 1week, 0.2ml acetone containing 5μg TPA was applied once a week for 20weeks. We used acetone as a vehicle to dissolve DMBA or TPA and to enhance the topical administrations. The control mice were similarly treated with the equal volume of acetone. The animals were lightly sedated using an intraperitoneal injection of ketamine/xylazine to immobilize the mice immediately before LEBS measurements. The measurement area was shaved using a clipper and a depilatory cream. We used the experimental setup shown in Fig. 2, in which the illumination beam size was 4mm in diameter. To calculate the LEBS enhancement factor without being affected by the hemoglobin absorption, we averaged the spectral signals from 615to635nm. In each mouse, we obtained four LEBS readings from different dorsal areas covering an area of 1.5×1.5in. We calculated a mean value of the LEBS enhancement factors from the four different readings in each mouse. We obtained sequential LEBS readings in vivo at 6 and 10weeks after the carcinogenesis initiation (i.e., DMBA treatment).

Result and Discussion of the Pilot Animal Study

We first evaluated all the mice in our pilot study by visual inspection. We confirmed that all carcinogen-treated mice used in this pilot study formed palpable papillomas of 1mm in diameter at 14weeks after the DMBA treatment (except the mouse used for histological evaluation), and the papillomas eventually progressed to invasive tumors. There were no papilloma formations at 10weeks after the first DMBA treatment. The histologic evaluations from one treated mouse at 10weeks showed dermal inflammation with overlying epidermal erosions or mild epidermal hyperplasia as a preneoplastic stage. Figure 7 shows that the LEBS enhancement factor at 10weeks is associated with cancer progression. On the other hand, at 6weeks, there was no significant difference in the LEBS enhancement factor between the carcinogen-treated and control mice. After 4weeks (i.e., at 10weeks), the LEBS enhancement factor revealed a statistically significant difference with the two-tailed pvalue=0.0135. The LEBS enhancement factor in the control group increased as the mice aged, while the carcinogen-treatment suppressed its increase. The distinct reduction in the LEBS enhancement factor of the treated mice at 10weeks suggests an alteration in the p(rls*) possibly due to changes in either ls* or structure details of the mice skin. Since the in vivo studies enabled us to monitor each animal at several different time points and to analyze the data specifically from the animals eventually harboring papillomas, our result was directly correlated with cancer progression.

Grahic Jump LocationF7 :

LEBS enhancement factor obtained from the precancerous skin tissue at 6 and 10weeks after the initiation of skin carcinogenesis as compared with the age-matched control mice. The LEBS enhancement factors do not show a significant difference between the carcinogen-treated mice and the control mice at 6weeks. However, the LEBS enhancement factors at 10weeks are statistically distinct. We confirmed that all the treated mice in this cohort formed palpable papillomas at 14weeks after the DMBA treatment.

The reduction in the LEBS enhancement factor in the carcinogen-treated group can be understood as follows. The mouse skin consists of three major layers: the epidermis, the dermis, and the hypodermis.27 The thickness of the mouse epidermis is only around 10μm, while the thickness of the female mouse dermis is approximately 200μm (Ref. 27). Because the LEBS enhancement factor contains light scattering signals mainly originating14 from tissue depth of Lsc(=120μm), the LEBS enhancement factor should contain the coherent partial waves that travel to the upper layer of the dermis. Recent studies about skin cancer also suggest that squamous cell carcinoma leads to decreased scattering power in the dermis probably due to degradation of collagen crosslinks.2829 Similar findings were observed in other types of epithelia cancers such as oral and cervical cancers.3032 Our result is consistent with these studies, although our study time points are at a stage of preneoplasia. Furthermore, our finding that the local scattering power increased with age in the control mice is also in agreement with the studies in cervical cancer.33 The previous animal studies12,1415 of precancerous alterations in colon cancer focused on the utility of the spectral properties of LEBS. On the other hand, our current study reports that a different quantification of LEBS (i.e., the enhancement factor) has the potential as an easily measurable robust biomarker, not dependent on the illumination intensity. Overall, our result supports the idea that the alterations in the LEBS enhancement factor can precede the development of papillomas (i.e., the classical early marker of skin carcinogenesis), and that the LEBS measurements potentially enable a better understanding of early carcinogenesis and prediction of cancer progression.

A drawback of our studies is that the exact physical origin of the change in the LEBS enhancement factor in our animal study was not clearly elucidated by the tissue phantom studies. This is mainly because measuring ls* in biological media is not trivial. For example, EBS measurements under coherent illumination are commonly used for measuring ls* in nonbiological tissue such as strongly scattering materials, given that the full width at half maximum (FWHM) of the EBS peak λ(3πls*). However, in the mouse skin, ls*0.5to1.0mm (Refs. 1,5) and thus the FWHM of the EBS peak is 0.003to0.007deg at 600nm. Measuring such an extremely narrow peak is not experimentally simple. As a result, EBS measurements are not commonly used to estimate ls* in biological tissue. We also attempted to measure ls* in the mice skin using conventional EBS measurements. However, due to this experimental challenge, the multiple EBS measurements from the same animal skin sample showed a 30% standard deviation in the EBS peak width. Thus, it was impossible for us to draw any solid conclusion that the change in the LEBS enhancement factor was originated from a change in ls*. Indeed, other methods such as integrating sphere measurements only permit rough estimation of ls* with large experimental deviations.1 In this respect, it is beyond the scope of our current study to report the exact physical origin that determines the LEBS enhancement factor in our animal study. Another limitation of our study is that we did not include any mathematical models that can describe the relationship between the LEBS enhancement factor and the sample optical properties.

We experimentally studied the properties of the LEBS enhancement factor using two complementary methods: (1) low-spatial-coherence illumination and (2) multiple spatially independent detections. The second configuration can be useful for studying angular dependency of light scattered from the sample over different spatial locations. We demonstrated that the intensity within the spatial coherence area can slightly change due to changes in either the macroscopic optical properties, or the detailed subtle structural features such as the relative refractive indices and the scatterer sizes. In particular, when Lsc<ls*, which is valid for most biological tissue, the LEBS enhancement factor can capture such alterations. Thus, the LEBS enhancement factor has potential as an easily measurable biomarker in skin carcinogenesis.

This project was supported in part by an American Cancer Society Institutional Research Grant No. IRG-58-006-47 to the Purdue Cancer Center, the ITRAC internal pilot grant mechanism of the Indiana University Simon Cancer Center, and a grant from Purdue Research Foundation. We thank Prof. Prabhakar Pradhan for the valuable discussion about our manuscript. We also thank Prof. Curtis Ashendel for helpful discussion about our animal study.

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Liu  Y., , Brand  R. E., , Turzhitsky  V., , Kim  Y. L., , Roy  H. K., , Hasabou  N., , Sturgis  C., , Shah  D., , Hall  C., , and Backman  V., “ Optical markers in duodenal mucosa predict the presence of pancreatic cancer. ,” Clin. Cancer Res..  1078-0432 13, , 4392–4399  ((2007)).
Woodworth  C. D., , Michael  E., , Smith  L., , Vijayachandra  K., , Glick  A., , Hennings  H., , and Yuspa  S. H., “ Strain-dependent differences in malignant conversion of mouse skin tumors is an inherent property of the epidermal keratinocyte. ,” Carcinogenesis.  0143-3334 25, (9 ), 1771–1778  ((2004)).
Azzi  L., , El-Alfy  M., , Martel  C., , and Labrie  F., “ Gender differences in mouse skin morphology and specific effects of sex steroids and dehydroepiandrosterone. ,” J. Invest. Dermatol..  0022-202X 124, (1 ), 22–27  ((2005)).
Salomatina  E., , Jiang  B., , Novak  J., , and Yaroslavsky  A. N., “ Optical properties of normal and cancerous human skin in the visible and near-infrared spectral range. ,” J. Biomed. Opt..  1083-3668 11, (6 ), 064026  ((2006)).
Brancaleon  L., , Durkin  A. J., , Tu  J. H., , Menaker  G., , Fallon  J. D., , and Kollias  N., “ In vivo fluorescence spectroscopy of nonmelanoma skin cancer. ,” Photochem. Photobiol..  0031-8655 73, (2 ), 178–183  ((2001)).
Pavlova  I., , Sokolov  K., , Drezek  R., , Malpica  A., , Follen  M., , and Richards-Kortum  R., “ Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy. ,” Photochem. Photobiol..  0031-8655 77, (5 ), 550–555  ((2003)).
Collier  T., , Arifler  D., , Malpica  A., , Follen  M., , and Richards-Kortum  R., “ Determination of epithelial tissue scattering coefficient using confocal microscopy. ,” IEEE J. Sel. Top. Quantum Electron..  1077-260X 9, (2 ), 307–313  ((2003)).
Wilder-Smith  P., , Osann  K., , Hanna  N., , El Abbadi  N., , Brenner  M., , Messadi  D., , and Krasieva  T., “ In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy. ,” Lasers Surg. Med..  0196-8092 35, (2 ), 96–103  ((2004)).
Brookner  C. K., , Follen  M., , Boiko  I., , Galvan  J., , Thomsen  S., , Malpica  A., , Suzuki  S., , Lotan  R., , and Richards-Kortum  R., “ Autofluorescence patterns in short-term cultures of normal cervical tissue. ,” Photochem. Photobiol..  0031-8655 71, (6 ), 730–736  ((2000)).
© 2010 Society of Photo-Optical Instrumentation Engineers

Citation

Jingjing Liu ; Zhengbin Xu ; Qinghai Song ; Raymond L. Konger and Young L. Kim
"Enhancement factor in low-coherence enhanced backscattering and its applications for characterizing experimental skin carcinogenesis", J. Biomed. Opt. 15(3), 037011 (June 09, 2010). ; http://dx.doi.org/10.1117/1.3443795


Figures

Grahic Jump LocationF1 :

Schematic illustration of the instance in which the LEBS enhancement factor can be distinct from the EBS enhancement factor under spatially coherent illumination. The two media [sketched in (a) and (d)] consist of scatterers of different relative refractive indices nr, different diameters Dp, and different numbers of particles (per milliliter), while the transport mean free path ls* and the anisotropic factor g are identical. In this case, (b) and (e) show the identical angular profiles with the enhancement factor of 2 because of the same ls*. On the other hand, the LEBS enhancement factors in the two media can be different, because the relative small coherent volumes determined by low-spatial-coherence illumination partially preserves the local optical properties as compared with the conventional measurement in the large volume.

Grahic Jump LocationF2 :

Complementary experimental setups using (a) EBS measurements under low-spatial-coherence illumination (i.e., LEBS), (b) multiple spatially independent detections using a microlens array, and (c) LEBS spectroscopy instrument. (a) The aperture A1 determines the spatial coherence length of illumination. A single lens L projects the angular distribution of the backscattering light onto the CCD camera. (b) The microlens array in the detection arm offers multiple mutually independent spatial filters under spatially coherent illumination. (c) The lens in the detection arm projects the angular distribution of the backscattered light onto the slit of the spectrograph, which disperses the light according to its wavelength in the direction perpendicular to the slit. The CCD records an intensity matrix as a function of the wavelength and the backscattering angle. P1 and P2 are polarizers. A1 and A2 are apertures. F is an interference filter.

Grahic Jump LocationF3 :

Angular profiles obtained from the water suspension of polystyrene microspheres of ls*=400μm and ls*=700μm under two different values of Lsc=115μm and Lsc=200μm. Because ls*Lsc3.5 in the both cases, the enhancement factors are statistically the same.

Grahic Jump LocationF4 :

(a) Enhancement factor as a function of the unitless length scale Lscls* measured from the water suspension of polystyrene microspheres (nr=1.19) and (b) enhancement factor over the unitless length scale Lscls*<1 for three different nr and Dp. The LEBS enhancement factor depends on subtle perturbations of ls*, nr, or Dp.

Grahic Jump LocationF5 :

Typical 3×3 angular profiles measured under spatially coherent illumination (i.e., He–Ne laser) using the microlens array that allows multiple spatially independent detections. The angular range of each EBS cone is from 3to3deg. The EBS cones are normalized by the intensity at a large scattering angle. The pitch of each microlens is 250μm.

Grahic Jump LocationF6 :

Enhancement factor matrix measured using the microlens array under spatially coherent illumination (i.e., Lscls*). Although the two media have the identical macroscopic scattering property (ls*=370μm and g=0.91 at 543nm), the multiple spatially independent detections reveal the influence of nr and Dp on the enhancement factor matrix.

Grahic Jump LocationF7 :

LEBS enhancement factor obtained from the precancerous skin tissue at 6 and 10weeks after the initiation of skin carcinogenesis as compared with the age-matched control mice. The LEBS enhancement factors do not show a significant difference between the carcinogen-treated mice and the control mice at 6weeks. However, the LEBS enhancement factors at 10weeks are statistically distinct. We confirmed that all the treated mice in this cohort formed palpable papillomas at 14weeks after the DMBA treatment.

Tables

Table Grahic Jump Location
Representative sample preparations of the scattering media of ls*=400μm and g=0.91.

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Woodworth  C. D., , Michael  E., , Smith  L., , Vijayachandra  K., , Glick  A., , Hennings  H., , and Yuspa  S. H., “ Strain-dependent differences in malignant conversion of mouse skin tumors is an inherent property of the epidermal keratinocyte. ,” Carcinogenesis.  0143-3334 25, (9 ), 1771–1778  ((2004)).
Azzi  L., , El-Alfy  M., , Martel  C., , and Labrie  F., “ Gender differences in mouse skin morphology and specific effects of sex steroids and dehydroepiandrosterone. ,” J. Invest. Dermatol..  0022-202X 124, (1 ), 22–27  ((2005)).
Salomatina  E., , Jiang  B., , Novak  J., , and Yaroslavsky  A. N., “ Optical properties of normal and cancerous human skin in the visible and near-infrared spectral range. ,” J. Biomed. Opt..  1083-3668 11, (6 ), 064026  ((2006)).
Brancaleon  L., , Durkin  A. J., , Tu  J. H., , Menaker  G., , Fallon  J. D., , and Kollias  N., “ In vivo fluorescence spectroscopy of nonmelanoma skin cancer. ,” Photochem. Photobiol..  0031-8655 73, (2 ), 178–183  ((2001)).
Pavlova  I., , Sokolov  K., , Drezek  R., , Malpica  A., , Follen  M., , and Richards-Kortum  R., “ Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy. ,” Photochem. Photobiol..  0031-8655 77, (5 ), 550–555  ((2003)).
Collier  T., , Arifler  D., , Malpica  A., , Follen  M., , and Richards-Kortum  R., “ Determination of epithelial tissue scattering coefficient using confocal microscopy. ,” IEEE J. Sel. Top. Quantum Electron..  1077-260X 9, (2 ), 307–313  ((2003)).
Wilder-Smith  P., , Osann  K., , Hanna  N., , El Abbadi  N., , Brenner  M., , Messadi  D., , and Krasieva  T., “ In vivo multiphoton fluorescence imaging: A novel approach to oral malignancy. ,” Lasers Surg. Med..  0196-8092 35, (2 ), 96–103  ((2004)).
Brookner  C. K., , Follen  M., , Boiko  I., , Galvan  J., , Thomsen  S., , Malpica  A., , Suzuki  S., , Lotan  R., , and Richards-Kortum  R., “ Autofluorescence patterns in short-term cultures of normal cervical tissue. ,” Photochem. Photobiol..  0031-8655 71, (6 ), 730–736  ((2000)).

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