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Abstract
Investigating the deformation of tissue architecture is one of the most important clinical methods for cancer diagnosis. Optical methods are now widely developed for rapid, precise, and real-time assessment of these alterations at the microscopic scale. One of the proposed methods is enhanced backscattering (EBS) technique that allows in-vivo measurement of the optical scattering characteristics. Here, EBS technique is employed to evaluate the optical anisotropy of human epithelial tissues as a measure to distinguish between normal and cancerous one. Orientation dependence of the mean scattering length is assessed in healthy and cancerous tissues of five different human organs i. e. uterus, bladder, colon, kidney, and liver. Helicity preserving channel and rotating ground glass diffuser are utilized to eliminate the polarization induced anisotropy and the background speckle noises respectively. Analysis of the backscattering cones recorded by a high-resolution CCD camera reveals the modification of the strength and degree of optical anisotropy in different tissues during cancer progression. Pathology data affirm the correlation between the experimental results and the morphological alteration of the epithelial cells in each carcinoma type. In general, tissues with fibrous constructional cells are subject to a decrease in anisotropy due to cancer, whereas those with cuboidal cells experience an increase in anisotropy. This complementary information enhances the potency of the EBS technique as a fast, non-destructive, and easily accessible tool for real-time tissue diagnosis.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Enhanced backscattering (EBS) technique measures angular dependent enhancement of the backscattered light due to the constructive interference of propagating photons in time-reversed paths through the scattering media. As the width of the angular distribution of backscattered intensity (called backscattering cone) strongly depends on the scattering strength of the disordered medium, the method has been employed to characterize the structural alteration of different scattering systems on the cellular and sub-cellular scale such as disordered materials [1], laser-cooled atoms [2], liquid crystals [3], photonics crystals [4], amplifying materials [5,6], and the biological tissues [7]. EBS method was first utilized in biological tissue studies by Yoo et al in 1990 for characterization of breast and lung cancers [8]. It was shown that the scattering mean free path of 620 nm laser light is longer in normal tissues than the cancerous one. Yoon et al in 1993 reported similar results for human forearm and chicken breast [9]. In 2006 Kim et al demonstrated the potential of EBS spectroscopy to determine the risk of colon carcinogenesis and colonoscopy-free screening for colorectal cancer [10]. They modified conventional setup using low coherent light source to reduce background speckle noise and achieving depth resolved information. In all the aforementioned reports EBS event has been traced for along a defined direction of light propagation in biological tissues so that only a certain cross-section of the backscattering cone has been considered as intensity profile. In fact, it has been assumed that the given biological tissues are optically isotropic such that backscattering cone is spatially symmetric. Nevertheless, there are few reports on anisotropic scattering of light in certain biological tissues [11–13]. Marquez et al utilized oblique incidence reflectometry to measure the absorption and the reduced-scattering coefficients of chicken breast tissue in the visible range (400 - 800 nm) [11]. They revealed the dependence of optical properties to the angle of fiber probe orientation. Kienle et al. demonstrated that the isointensity contours of reflectance profile (R(x, y)) are elliptical for some optically anisotropic biological tissues (e.g., muscle, skin, bone, and tooth) that have aligned myofibrils and collagen fibers [12]. Ushakova et al have demonstrated the anisotropy of light scattering in demineralized bone using laser video-reflectometry and EBS technique [13]. To our knowledge, no study has been conducted on the assessment of optical anisotropy of biological tissues during cancer progress. Here, EBS technique is employed to investigate the anisotropy regarding the light scattering in healthy and cancerous tissues of five different human organs i. e. uterus, bladder, colon, kidney and liver. The assessment appends additional information to the typical EBS data, which enhances the capability of technique as a fast, non-destructive and easy accessible tool for real-time tissue diagnosis.
2. Optical scattering in epithelial tissues
Mean value of the scattering length is an approximate quantity to show the cumulative effect of multiple scattering events in a heterogeneous or even anisotropic medium corresponding to the real scattering length, ls(x, y, z, t), that is a function of position and time. The reason for is that no simple, fast and accurate method has been developed to determine the functional form of the optical quantities in a given disordered medium. This mean value can be accurately introduced as an indicator to monitor the alterations in given media.
2.1 Mean scattering length
Figure 1 illustrates simple model of light scattering in tissues with different architectures. In Fig. 1(a), large number of small basic cells with large black color nuclei (indicating high RI contrast of the nucleus with its environment) provide successive scattering events for incident light which results in small mean scattering length ls. On the other hand, photons propagating within medium containing relatively small number of larger basic cells with small grey nuclei (resembling low RI contrast tissue) face fewer scattering events as demonstrated in Fig. 1(b). In the latter case, mean scattering length increases as the distances between scattering events elongate.
Fig. 1. Light scattering in epithelial tissues with different architectures (a) large number of small basic cells with large black color nuclei (high RI contrast) (b) relatively small number of larger basic cells with small grey nuclei (low RI contrast)
2.2 Optical anisotropy
The concept of anisotropy of light scattering in biological tissues is mainly refered to those whose nano/micro scale constitutive structures are anisotropic in shape (e.g., axons, myofibrils or collagen fibers) [11,12]. The scattering phase function of these microstructures is shown to be asymmetrical [14]. Single scattering phase function describes the angular distribution of the scattered light for a specific wavelength. As a result, the cumulative effect of light scattering from successive particles, called multiple scattering, would be dependent on the direction of light propagation in the tissue under study. In addition, anisotropy may even arises from the non-homogeneous distribution of isotropic-shape basic cells. Figure 2 depicts scattering of light in four media: (a) spherical scatterers (which are themselves optically isotropic) are homogeneously distributed in the medium. (b) Medium containing ordered cylindrical particles (three different pathways that photons may travel is also shown) (c) Medium that is optically anisotropic due to the heterogeneous distribution of the spherical scatterers and (d) isotropic medium consist of anisotropic cylindrical scatterers that are randomly oriented in space. It is clearly seen that in cases (b) and (c), the photons in the pathways perpendicular to the zx plane confront different heterogeneity compared to those in paths perpendicular to the yx plane. On the other hand, in (a) and (d), optical scattering parameters are spatially isotropic.
Fig. 2. (a) Scatterers are homogeneously distributed in the medium containing spherical scatterers. (b) Medium containing cylindrical particles and three different pathways that photons may travel. (c) Medium that is optically anisotropic due to the heterogeneous distribution of the spherical scatterers and (d) isotropic medium consist of anisotropic-shape cylindrical scatterers that are randomly oriented; Insets: typical cross sections of the media.
From the above example, it can be concluded that anisotropy in a medium strongly depends on the shape and distribution of its constituent cells.
More than 80% of the human cancers occur in tissues containing various types of polyhedral microstructures known as epithelial cells [15]. Epithelium has either simple (unilayered) or stratified (multi-layer) structure composed of micron-size epithelial cells. As demonstrated in Fig. 3(a), there are three major shapes of the epithelial cells in human organs: squamous (thin flat polygonal plates), cuboidal (cubic shaped), and columnar (taller than they are wide) [16]. In the case of epithelial tissues, anisotropic constituents are columnar cells. The aligned columnar cells have scattering properties similar to the nematic liquid crystal (NLC) molecules which become aligned under the applied magnetic fields [17].
Fig. 3. (a) Three major shapes of the human epithelial cells: squamous (thin flat polygonal plates), cuboidal (cubic shaped), and columnar (taller than they are wide) (b) drastic changes of the cell architecture during cancer progress.
In the stage of dysplasia, cell architecture and orientation undergo drastic changes as graphically represented in Fig. 3(b). In particular, normal cells become crowded, hyperchromatic, and suffer of nuclear enlargement [15].
Either scattering centers become larger or the number of scatterers increases, the scattering length will decrease. Moreover, as a result of the deformation of the cells and/or changing their orientation, the optical anisotropy of the medium may change. Depending on the type of basic cells, these variations may be different and our aim is to investigate these differences using EBS technique.
3. Enhanced backscattering
In enhanced backscattering phenomenon, light waves propagating time reversed paths in a disordered medium interfere constructively to produce enhanced scattered intensity in the backward direction [18–20]. Amount of enhancement depends on the distance which photons traverse before leaving the medium. The photons travelling short path length through the medium interfere over a wide range of angles, whereas very long paths contribute only in exact backscattering direction. Figure 4(a) shows simple graphical representation of EBS event as well as a typical cross section of backscattering cone in focal plane (x-y) perpendicular to the incident light direction (z). Based on diffusion theory for a monochromatic light propagating with wavelength${\; \lambda }$ in an isotropic scattering medium, the EBS cone profile is determined mainly by the transport mean free path length ls and has a full angular width at half-maximum of
regarding any direction in x-y plane [21–23].Fig. 4. (a) Schematics of EBS event. Picture in bottom shows typical cross section of backscattering cone in focal plane perpendicular to the incident light direction (b) Schematics of EBS setup. NDF: neutral density filter, P: linear polarizer, L: lens, BE: beam expander, BS: beam splitter.
Numerical simulations [14,17] as well as the experimental studies of disordered media containing aligned cylindrical microstructures with anisotropic polarizability such as nematic liquid crystals [24], etched gallium phosphide samples [25]and biological tissues having micro fibers such as muscle, skin, bone and tooth ascertain anisotropic diffusion of light [12,26]. As a result, it is expected that the optical anisotropy of the media causes the asymmetry of the backscattering cone, and therefore, the FWHM dependence on the direction of determination in x-y plane.
4. Materials and methods
The experimental setup for recording EBS cone is depicted in Fig. 4(b). CW diode laser with output power of 100 mW at the wavelength of 650 nm was used to illuminate the tissue samples. Laser wavelength is selected within the spectral range of the optical window of the human tissue in which the absorption from oxygenated blood, deoxygenated blood, skin and fatty tissue is lowest [27]. Laser power was controlled by a set of ND filters to select optimum illumination power. It is worth noting that in experiments on strong scattering media with coherent light source, the sample was usually rotated by its retaining platform to remove speckle noises [26,28]. While this effectively eliminates speckle pattern, it also ignores anisotropy of intensity distribution. Therefore, to measure the tissue’s anisotropy, a rotating ground glass diffuser (RGGD) driven by a stepper motor was utilized to efficiently reduce the speckle pattern as reported by Stangner et al [29]. Moreover, all recorded images were denoised using appropriate speckle reduction algorithm. Figure 5 demonstrates typical backscattered cone images of kidney tissue recorded by CCD camera, and the corresponding 2D and 3D contours before and after denoising. The laser beam was focused on GGD and scattered light was then collected by an objective to form low spatial coherent laser beam. Using a $\lambda /4$ polarizer plate, linear polarized laser beam was converted to circular polarized one. Using circular polarizers, EBS was measured in helicity preserving channel to prevent polarization induced anisotropy in the backscatter cone [25]. It also blocks the single scattering contribution which effectively reduces the background noise of the EBS cone to gain better enhancement factor [25]. Each single scattering event flips photon’s helicity while the multiple scattered photons lose their polarization state. Gaussian TEM00 mode was achieved utilizing a spatial filter and a proper iris and beam diameter of 8 mm was obtained after passing through a beam expander.
Fig. 5. (a), (d) typical backscattered cone images of normal kidney tissue recorded by CCD camera, and the corresponding (b), (e) 2D and (c), (f) 3D contours before and after denoising.
Half of the beam was reflected by a 50:50 beam splitter onto the tissue and the remaining was vanished in the beam dump. It is worth mentioning that the beam-splitter is used in divergent portion of the optical beam (in front of the rays scattered from the tissue). Hence, cube beam-splitter is not proper because it may contribute substantial amounts of unwanted aberrations. Accordingly, a super-flat non-polarizing, anti-reflection coated plate beam splitter was used to eliminate unwanted false images. The corresponding angular resolution was ∼ 10 µrad which is proper for studying the anisotropy of the backscattering cone image.
The tissue scattered laser to another quarter wave plate and linear polarizer. The backscattering image was collected by a convex lens and recorded using high resolution CCD (3840 × 2160 pixels, 30frame/second), placed exactly in the focal plane of the converging lens. In a semi-infinite scattering medium, an infinite number of time-reversed path-pairs with different spatial separations will combine to form the EBS angular intensity distribution [30] which peaks at the center of the exact backscattering direction and sufficiently away from this direction, the integrated signal vanishes. As such, the converging lens (L5) integrates the interference patterns from all possible sets of time reversed path-pairs. In the language of the Fourier optics, angular dependence of the EBS cone profile is a Fourier transform of the spatial intensity distribution of backscattered light [22].
Optical alignment was carefully accomplished to minimize the astigmatism and avoid any anisotropy due to the response of the setup. Tissue samples of 5 different patients who suffered of dysplastic transformation at the following organs: colon, bladder, kidney, liver and uterus were provided from department of Cancer Institute, Imam Khomeini medical Centre, Tehran. The protocol for the use of tissues was consented by the ethics committee of Imam Khomeini medical Centre. Tissue sections were kept in 10% neutral buffered formalin immediately after surgery (or biopsy) to prevent the unwanted deformation. The slices were cut in 10×10×8 mm dimensions just before the EBS experiments.
The cancerous samples were assigned as carcinoma in situ after biopsy. For each organ 8 samples containing 4 adjacent pieces of normal and 4 adjacent pieces of cancerous lesions were assessed so that both normal and infected tissues were taken from one patient. The normal tissue pieces were selected from the areas adequately far from the lesion margin to avoid the likelihood of measuring adjacent carcinoma in situ. Adjacent pieces have been examined after cutting without changing the initial position respect to each other.
5. Results and discussions
Figure 6 illustrates recorded EBS cones and the corresponding color contour graphs of typical normal and lesion pieces of five different tissue types. Notice that the original de-noised EBS cone images (such as EBS cone of normal kidney in Fig. 5) are so pale that it is not possible for the human eye to simply recognize their shape and corresponding anisotropy.
Fig. 6. Recorded EBS cones (after denoising) and the corresponding color contour graphs of typical normal and lesion pieces of five different tissue types: Colon, Bladder, Kidney, Liver and Uterus.
Therefore, the intensity profile was re-scaled to increase the intensity contrast benefit to enhance the image resolution. In addition, in some cases (e. g. normal uterus, normal bladder and cancerous liver) in which the calculated mean scattering length showed heterogeneity and/or the measured FWHM was relatively small, the image of the cone’s top was zoomed to better demonstration of the heterogeneity. Instead, those images in Fig. 6 look like to be saturated which is not the case. It should be emphasized that such modified images were provided only to compare the cone shapes qualitatively not for the calculation of the real FWHM.
For each EBS cone, FWHM was determined along the four directions i.e. 0°, 45°, 90° and 135°, and the corresponding mean scattering lengths ls were calculated using Eq. (1). Table 1 summarizes the values of measured ls in normal pieces as well as the lesions of colon, bladder, kidney, liver and uterus tissues regarding different directions of light propagation. Notice that angle ϕ=0° is allocated to the light propagation direction with maximum value of ls.
Table 1. Scattering length ls for normal pieces (N) and lesions (L) of colon, bladder, kidney, liver and uterus tissues regarding the angular directions: 0°, 45°, 90° and 135° (Note: angle ϕ=0° is allocated to the light propagation direction with maximum value of ls).
The light scattering lengths ls in normal and cancerous samples were statistically analyzed by means of the standard paired t-test based on Shapiro-Wilk normality test in SigmaPlot v.14 package. Assessment revealed that in general, the maximum value of ls is larger than the normal one at the 95% confidence level. This indicates that the increase in the ls parameter of cancerous kidney tissue compared to the normal one is a rare event. The difference in the scattering length value of normal and cancerous tissues illustrated in Fig. 7 supports this conclusion. Figure 7 shows maximum difference between mean values of the scattering length, ls,avg (averaged over different directions) for five tissue types based on the data presented in Table 1. In general, it is seen that ls,avg reduces during the cancer progress in all the tissue types except the kidney.
In fact by starting mutations in cells, colonization occurs in infected organ which causes the scattering centers to be increased, thus, the scattering length is expected to be reduced. This event occurs in all organs except the kidney in which the scattering length is increased. Reduction of the scattering length in kidney tissue indicates that there is not necessarily an increase in the scattering strength for all organ tissues during the cancer as explained in several reports [9, 10 and 15].
Histological assessment of the tissues revealed that this can be explained by the way kidney getting cancerous. According to our pathology data, type of each carcinoma is specified as follows: leiomyosarcoma in uterus, urothelial carcinoma in bladder, adenocarcinoma in rectal (poorly differentiated with signet ring cells), fibrolamellar hepatocellular carcinoma in liver and clear cell renal cell carcinoma (CCRCC) in kidney. It is known that the CCRCC shows different morphological alteration than the other cancerous types [31]. In microscopic scale, the tumor cells of CCRCC are usually cuboidal with a centrally or basally located nucleus. The clear cell form of the tumor cells results from the reposition of glycogen and lipids due to irregularities of carbohydrate metabolism [31]. This makes the tissue being optically more uniform because of the local accumulation of cytoplasm in different parts of the tissue, as depicted in histology images of Fig. 8. As a result, the spatial contrast of the dielectric constant value in kidney tissue is expected to be decreased with progression of the CCRCC cancer leading to reduction of the scattering strength and elongation of the mean scattering length (see Fig. 1(b)).
Fig. 8. Histology images of kidney lesion tissue (CCRCC type cancer) (a) H&E stain (b) PAX8 stain and (c) CK AE1/3 stain.
As demonstrated in Fig. 9(a), liver tissue with the smallest scattering strength (the largest scattering length) in health, shows the highest increase in the scattering strength after dysplasia. It is noticeable that about 70–85% of the liver volume is occupied by parenchymal hepatocytes [32]. As a result, the scattering strength of the healthy liver tissue is expected to be lower than the other ones. On the other hands, liver biopsy shows heterogeneous sheets, nests, and trabeculae of tumor cells separated by dense collagen bundles. The large polygonal tumor cells have well-defined borders and eosinophilic, coarsely granular cytoplasm with large vesicular nuclei [33]. These heterogeneities lead to enhance the refractive index contrast in this tissue, which results in increasing the number of scattering events and, consequently, a significant decrease in scattering length (see Fig. 1(a)).
Fig. 9. (a) Maximum and (b) mean values of the scattering length (averaged over different directions) for five tissue types based on the Table 1.
In order to investigate the optical anisotropy, angular variation of the scattering length ls for normal and cancerous tissues of colon, bladder, kidney, liver and uterus organs was assessed as revealed in Fig. 9. While the scattering length value is spatially isotropic for normal kidney it significantly varies against the angular orientation of light propagation in other organs. The reason is the simple cuboidal form of the epithelial cells in healthy kidney rather than the columnar cells in other tissue types (see Table 1). It has already been reported that the disordered materials having anisotropic-shape microstructures show anisotropy in optical transport mean free path [3,23,34–36].
Furthermore, anisotropy of scattering strength slightly increases in the case of the cancerous kidney respect to the normal one which is mainly due to the proliferation of the fibrous in kidney tissue after become cancerous (see Fig. 8). According to Fig. 10, anisotropy strength (defined as difference between the maximum and minimum values of the scattering length over different directions) are determined.
Fig. 10. Angular variation of the scattering length ls for normal and cancerous tissues (lesions) of colon, bladder, kidney, liver and uterus organs.
The results for different examined tissues have been compared in Fig. 11 in which three different events can be recognized as follows: (i) in liver, bladder and uterus tissues: anisotropy decreases (ii) in colon and kidney tissues: anisotropy increases.
Fig. 11. Anisotropy strength (difference between the maximum and minimum values of scattering length over different directions) for healthy and cancerous tissues.
Regarding the event (i), relatively lower degree of anisotropy in cancerous tissues ascertains more randomness of the spatial orientation of columnar cells after dysplasia so that the cumulative effect on the photon propagation is that the scattering strength loses dependence on the spatial direction as can be seen in Fig. 2(b) and (d). On the other hand, what is happened for colon and kidney tissues (event (ii)) is in contrary to that of uterus, bladder and liver. As demonstrated in Fig. 11, colon and kidney tissues have been exposed to increased anisotropy. Figure 12 shows histology images of (a) uterus (Leiomyosarcoma), kidney (CCRCC) and colon tissues (Signet ring cell adenocarcinoma).
Fig. 12. Histology images of (a) uterus (Leiomyosarcoma), (b) kidney (CCRCC) and (c) colon tissues (Signet ring cell adenocarcinoma)
In the case of uterus cancer (Fig. 12(a)), unlike the kidney, there was no increase and accumulation of cytoplasm but cellular atypia and numerous mitoses. Here, order of orientation of the tall columnar cells has been somewhat lost and replaced with the randomness, leading to decreased anisotropy (similar to example shown in Fig. 2(d)). However, in Fig. 12(b) and (c), deformed cells with small nuclei and increased cytoplasm, as well as the signet ring-shaped cells with marginal nuclei, reinforce anisotropy. Table 2 summarizes the results by ranking the tissue types based on the amount of changes in scattering strength and anisotropy. In general, tissues with columnar epithelial cells or Hepatocytes (in the case of liver) exhibit more alterations respect to the other ones.
Table 2. Scattering length ls for normal pieces (N) and lesions (L) of colon, bladder, kidney, liver and uterus tissues regarding the angular directions: 0°, 45°, 90° and 135°
6. Conclusion
The aim of this paper was to investigate the evolution of the optical anisotropy of epithelial tissues during several types of cancer. EBS technique with technical modifications i. e. utilizing helicity preserving channel (to eliminate the polarization induced anisotropy) and rotating ground glass diffuser (to suppress the background speckle noise) has been employed to measure the scattering length of the laser beam along different directions of propagation in biological tissues. Analysis of the backscattering cones recorded by a high resolution CCD camera reveals that not only scattering strength but also spatial anisotropy of the scattering length modifies in different tissues during cancer progress. Pathology data ascertain that these variations are strongly correlated to the shape of the epithelial cells and their morphological alteration during each carcinoma type. In general, the tissues with fibrous stem cells are subject to a decrease in anisotropy due to cancer, whereas those with cuboidal cells experience an increase in anisotropy. Further study is currently underway to evaluate the proposed analysis for tissue diagnosis in different stages of cancer progress using depth resolved low coherent EBS technique. We hope that the findings open up a new approach to diagnose biological tissues especially in the case of cancer in early stages.
Disclosures
The authors declare no conflicts of interest.
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