We report the development and implementation of a unique integrated Mueller-matrix (MM) near-infrared (NIR) imaging and Mueller-matrix point-wise diffuse reflectance (DR) spectroscopy technique for improving colonic cancer detection and diagnosis. Point-wise MM DR spectra can be acquired from any suspicious tissue areas indicated by MM imaging. A total of 30 paired colonic tissue specimens (normal vs. cancer) were measured using the integrated MM imaging and point-wise MM DR spectroscopy system. Polar decomposition algorithms are employed on the acquired images and spectra to derive three polarization metrics including depolarization, diattentuation and retardance for colonic tissue characterization. The decomposition results show that tissue depolarization and retardance are significantly decreased (p<0.001, paired 2-sided Student’s t-test, n = 30); while the tissue diattentuation is significantly increased (p<0.001, paired 2-sided Student’s t-test, n = 30) associated with colonic cancer. Further partial least squares discriminant analysis (PLS-DA) and leave-one tissue site-out, cross validation (LOSCV) show that the combination of the three polarization metrics provide the best diagnostic accuracy of 95.0% (sensitivity: 93.3%, and specificity: 96.7%) compared to either of the three polarization metrics (sensitivities of 93.3%, 83.3%, and 80.0%; and specificities of 90.0%, 96.7%, and 80.0%, respectively, for the depolarization, diattentuation and retardance metrics) for colonic cancer detection. This work suggests that the integrated MM NIR imaging and point-wise MM NIR diffuse reflectance spectroscopy has the potential to improve the early detection and diagnosis of malignant lesions in the colon.
© 2016 Optical Society of America
Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in males and the second in females worldwide, with an estimated 1.4 million cases and 693,900 deaths occurring in 2012 . In Singapore, CRC has become the most frequent cancer with a total of 9,324 new cases diagnosed from 2010 to 2014 . Current routine screening of CRC uses white light reflectance (WLR) colonoscopy which may reduce CRC incidence and mortality . However, some individuals are still diagnosed with CRC despite recent colonoscopy . This is probably because conventional WLR colonoscopy heavily relies on the visualization of gross mucosal features associated with neoplastic transformation . Subtle tissue changes may not be apparent, limiting its diagnostic accuracy. Consequently, existing diagnostic guidelines recommend extensive but random biopsy samplings during colonoscopic inspections of patients , followed by the microscopic examination which is highly subjective and depends heavily on the experiences of the pathologists. Overall, the current approach for colonic tissue diagnosis is clinically labor intensive and a burden to the patients. There is a need to develop advanced optical diagnostic techniques for objective diagnosis and characterization of colonic tissue.
Polarized light imaging/spectroscopy has been comprehensively investigated for tissue diagnosis [6–17]. Polarized light implementation offers several compelling advantages as follows: (i) surface and beneath-the-surface detection of biological tissue taken from the tissue depolarization properties [11, 15]; (ii) tissue anisotropy analysis through the tissue diattentuation and retardance [13, 14]; (3) enhanced tissue diagnosis through the combination of complementary depolarization, diattentuation and retardance of the tissues . Among the various polarized light imaging/spectroscopy techniques developed [6–14, 18], Mueller-Matrix polarimetry is capable of measuring the complete polarimetric transfer function [6–9], known as Mueller-matrix, of the bulk biological tissues which are optically inhomogeneous, birefringent, and absorbing media . Currently, biomedical Mueller-Matrix polarimetry is mostly centered on the use of short visible wavelengths of illumination light that has a limited penetration depth and cannot detect lesions in deeper areas [8,9]. The near-infrared (NIR) light, on the other hand, penetrates much deeper into the tissue, and is well suited for deep tissue diagnosis [12, 20–22]. Further, the reported Mueller-Matrix polarimetries only acquired either the images [7,8] or the optical spectra  of the biological tissues alone. In this work, we report on the development of a unique integrated Mueller-Matrix NIR imaging and point-wise Mueller-Matrix spectroscopy system for colonic tissue diagnosis and characterization. Point-wise Mueller-Matrix spectra can be acquired from any suspicious tissue areas indicated by Mueller-Matrix imaging. Polar decomposition algorithms are employed on the acquired Mueller-Matrix images/spectra to derive three polarization metrics including depolarization, diattentuation and retardance. Partial least squares discriminant analysis (PLS-DA) and leave-one tissue site-out, cross-validation (LOSCV) are implemented on the derived spectroscopic polarization metrics (i.e., depolarization, diattentuation and retardance) to develop robust spectral diagnostic models for the differentiation between cancerous and normal colonic tissues.
2. Materials and methods
2.1 Integrated Mueller-matrix NIR imaging and point-wise diffuse reflectance spectroscopy system
Figure 1 shows the schematic of the integrated Mueller-Matrix NIR imaging and point-wise diffuse reflectance (DR) spectroscopy system developed for tissue measurements. The light from a tungsten halogen lamp (HL-2000, Ocean Optics Inc., Dunedin, FL) is coupled into a 200 fiber and passes through a beam expander, a long-pass filter (FEL0700, Thorlabs, Newton, NJ), a polarizer (LPNIR100-MP2, Thorlabs, Newton, NJ), and a quarter waveplate (AQWP10M-980, Thorlabs, Newton, NJ) for tissue illumination. The NIR diffuse reflectance photons backscattered from the tissue pass through a quarter waveplate (AQWP10M-980, Thorlabs, Newton, NJ), a polarizer (LPNIR100-MP2, Thorlabs, Newton, NJ), a collection lens, and a specially designed point-spectrum optical adaptor  before detected by a CCD camera (Pixis 1024, Princeton Instruments, Trenton, NJ). The customized optical adaptor comprises three lenses (f = 50 mm), a thin quartz glass plate (25 × 25 × 1 mm3) coated with a gold mirror (diameter of 100 µm, reflection of ~99% in 700-1100 nm) and a 2-D motorized translational stage. During tissue measurement, a small portion of the backscattered light was reflected by the optical adaptor and collected by a spectrometer (QE65000, Ocean Optics Inc., Dunedin, FL) for tissue spectroscopic analysis .
To acquire the 4 by 4 Mueller-Matrix DR images/spectra, the fast axis of the polarizers (P1, P2) is fixed while the quarter waveplates (QWP1, QWP2) were rotated automatically by using two pairs of gears and two sets of step motors in the excitation and collection paths, respectively. The rotation speed ratio of the two quarter waveplates is fixed at 1:5. The detected intensity is Fourier modulated as follows [24, 25]:Fig. 1), t is the exposure time of the camera, and a0, an, bn are the Fourier coefficients which can be derived through the detected intensity I. With the integrated Mueller-Matrix NIR imaging and point-wise spectroscopy system developed, a set of 25 Mueller-Matrix images/spectra can be acquired from colonic tissues in tandem within 5 s; hence the 4 by 4 Mueller-Matrix imaging/point-wise spectroscopy can be generated for tissue diagnosis and characterization. Further automatic motorization of the small gold mirror coated on the quartz plate together with the point-wise spectral measurement module enables a rapid movement of the dark spot (of 0.2 mm in diameter due to the reflection of gold mirror in the point spectrum optical adapter) to any spot of the imaged tissue of interest on the Mueller-Matrix image, and the subsequent 4 by 4 Mueller-Matrix point-wise spectroscopy can be realized within 1 s. One notes that the Eq. (1) applies only when the retarders used are quarter waveplates, and their angular speed ratio is fixed at 1:5. More general formalism for Mueller-Matrix measurements with dual rotating retarders rotating with different speed ratios can be found in .
To derive the colonic tissue polarization metrics (i.e., diattentuation D, depolarization Δ, and retardance R), polar decomposition  is implemented on the 4 × 4 Mueller-Matrix images/spectra acquired with the system developed (Fig. 1). Briefly, the tissue Mueller-Matrix M is expressed as the product of three 4 by 4 matrices: the diattentuation matrix (MD), the depolarization matrix (MΔ), and the retardance matrix (MR) :
The diattentuation vector is determined by the elements on the first row of the Mueller-Matrix :
The diattentuation, D, and the diattentuation matrix, MD, can thus be determined as :
Note that has no diattentuation, and it can be further decomposed as a retardance matrix (MR) followed by the depolarization matrix (MΔ) :
If the determinant of is negative, the minus sign is applied. Otherwise, the plus sign is applied .
Using Eqs. (8) and (9), MΔ can be determined, and MR can be obtained by:
Finally, the depolarization Δ, and retardance R can be calculated as follows:
To validate the performances of the system developed, the Mueller-Matrix NIR spectra of a half waveplate and a quarter waveplate were measured and decomposed. The difference between the measured retardance and that provided by the manufacturer is less than 3%, confirming the robustness of the system developed.
2.2. Statistical analysis
The unpaired two-sided Student’s t-test was used to evaluate the decomposed Mueller-Matrix spectroscopic differences between cancer and normal colonic tissues . Partial least squares (PLS) - discriminant analysis (DA) was applied on the derived spectroscopic polarization metrics for developing spectral diagnosis models . Leave-one-tissue site out, cross-validation (LOSCV) was further used to assess and optimize the PLS-DA model complexity, while reducing the risk of over-fitting. The above multivariate statistical analysis was performed using in-house written scripts in the Matlab programming environment (Mathworks. Inc., Natick, MA).
2.3. Colonic tissue specimens
A total of 30 paired (i.e., normal vs cancer) colonic tissue specimens (average size of ~6 x 3 x 3 mm3) were collected from 30 patients (18 men and 12 women with a mean age of 56) who underwent partial colectomy or surgical resections with clinically suspicious lesions or histopathologically proven malignancies in the colon. All patients preoperatively signed an informed consent permitting the investigative use of the tissue, and this study was approved by the Institutional Review Board (IRB) of the National Healthcare Group (NHG) of Singapore. Immediately after surgical resections, tissue specimens were put into vials with physiological saline solution which were stored in a flask with ice (−4 °C) for sending to the Optical Bioimaging Laboratory within 10 minutes for Mueller-Matrix (MM) NIR imaging and point-wise spectroscopy measurements. The paired tissue specimens from each patient were placed on a quartz glass slide (26 × 76 × 1.2 mm3) (cancer tissue was placed at the lower part of the slide while the normal one was placed at upper part of the slide) for MM NIR imaging measurements. After the MM NIR imaging/spectra acquisitions, the tissue specimens were fixed in 10% formalin solution and then sent back to the hospital for histopathological examinations. The histopathological examinations confirmed that 30 tissue specimens were normal, and 30 tissue specimens were cancer (moderately differentiated adenocarcinoma). Figure 2 shows the NIR diffuse reflectance image of one typical paired colonic tissues (~6 x 3 x 3 mm3).
3. Results and discussion
With the integrated Mueller-Matrix NIR imaging and point-wise spectroscopy technique developed, we are able to acquire 4 by 4 Mueller-Matrix NIR images of 30 paired colonic tissues. Figure 3 shows the representative normalized Muller matrix images of the paired (normal vs. cancer) colonic tissue sample as confirmed by histological examinations. All the Mueller-Matrix elements (except m11) are normalized by m11. It is observed from Fig. 3 that the diagonal elements of the Mueller-Matrix are much higher than the non-diagonal elements, reflecting a high depolarization power of the colon tissue. Interestingly, m22 and m33 element are identical not only for normal colon tissues, but also for the cancerous colon tissues. Besides, the magnitude of m22 and m33 are much higher than that of m44, indicating that the backscattered light is less depolarized when the incident light is linearly rather than circularly polarized. The magnitudes of m22 and m33 are higher for colon cancer than normal colon tissue, indicating a lower depolarization power of colon cancer tissues. The results we observed are consistent with previous reports in literature [9, 17]. We also found the existence of non-diagonal Mueller-Matrix images (i.e., m34, and m43), demonstrating the anisotropy of colonic tissues. The colonic anisotropy might originate from NIR light propagation in birefringent collagen located in the submucosa layer of the colonic tissue . This is because the NIR light penetrates deeper (~1 mm) [12, 20–22] inside the colonic tissue, and the diffuse reflectance signal of the deeper collagen  can be detected and reflected in the non-diagonal Mueller-Matrix images.
Given the abundance of the diagnostic information contained in tissue Mueller-Matrix images acquired (Fig. 3), the quantitative biophysical polarization metrics (i.e., diattentuation, depolarization, and retardance) were derived using the polar decomposition algorithms  (Fig. 4). As shown in Fig. 4(a), the diattentuation of colonic cancer tissue is higher than that of normal colonic tissue. The diattentuation profile [Fig. 4(d)] confirms the significantly increased diattentuation for the colon cancer. One notes that biomolecules such as amino acids, proteins and nucleic acids exhibit diattentuation effects . The higher magnitude observed for the diattentuation of cancerous tissue compared to normal tissue may be due to the enlarged nuclei and increased concentrations of chromatin (hence, nucleic acids) during colonic cancer development , which led to the increase in diattentuation effects in colonic cancer. Further, the decomposed depolarization image confirms that the cancerous colon clearly exhibits less depolarization effects [Figs. 4(b), 4(e)]. The decreased depolarization of cancer tissue can be attributed to the multiple scattering effects of polarized incident light in the bulk colonic tissue, originating from variations in the refractive indices of microstructures in cancer tissue . Since an increase in cellular and nuclear sizes is accompanied with high cellular density and vascularization during cancer progression, an enhancement in anisotropic or Mie scattering (directionally dependent) of light in cancerous tissue could result in less depolarizing effects as compared to more isotropic or Rayleigh scattering in normal tissue [18, 31]. It is also observed that the decomposed retardance distributions are different between the normal and cancer colonic tissues. Overall, the very different diattentuation, depolarization and retardance images between normal and cancer colonic tissue observed (as shown in Fig. 4) demonstrate the potential of Mueller-matrix NIR imaging for early diagnosis and characterization of colonic cancer.
To determine the specific biochemical changes associated with colonic cancer, we have further acquired 60 sets (normal: n = 30; cancer: n = 30) of point-wise Mueller-matrix DR spectra from the suspicious tissue regions as indicated by the Mueller-Matrix images (Fig. 3). Figure 5 shows the typical 4 by 4 Mueller-Matrix DR spectra acquired from the histopathologically confirmed normal and cancerous colonic tissues. Obviously, the values of m22 and m33 are identical, which are consistent with Mueller-Matrix imaging (Fig. 3). Besides, the magnitude of spectroscopic m22 and m33 are higher in the colon cancer tissues, reconfirming the lower depolarizing power of cancer tissue. We also found non-diagonal spectroscopic Mueller-matrix elements (i.e., m34) to be non-zero, substantiating the anisotropy of colonic tissue. One notes that m11 generally represents the overall diffuse reflectance spectra of colonic tissue when unpolarized light is used . Further analysis conducted on m11 (Fig. 5) casts light on the biochemical changes associated with colonic cancer. Overall, the NIR (700-1100 nm) DR spectra of both the normal and cancerous colonic tissues are dominated by the characteristic of water (970 nm)  and hemoglobin (940 nm) absorption bands . Specifically, prominent water absorption valley is observed at 970 nm for both tissue types (Fig. 5). The water absorption band at 970 nm is due to the combination of the first harmonic of the O-H symmetric stretch vibration and the fundamental anti-symmetric stretch vibration from hydrogen bound O-H , making it a sensitive indicator of the local environment of water molecules . Further, we found the water absorption valley is more obvious on the cancer tissue than that on the normal one, indicating increased water content for the cancerous colonic tissues. The enhanced metabolic rate in colonic cancer  contributes to the increased water content as water provides the conversion of mechanical energy developed by contractile proteins into the chemical energy useful in cell process . The increased water for the cancerous colonic tissue has also been observed in other cancer tissues (e.g., esophagus , stomach , cervix [39–41] and brain ) by using Raman spectroscopy [4, 28, 38–42]. We have also found the decreased DR of hemoglobin band (940 nm) for the colonic cancer, signifying the increased hemoglobin content associated with colonic cancer tissue. The increased hemoglobin content for colonic cancer could be attributed to the increased microvasculatures in malignant tumors [36, 43]. The above observation is also consistent with previous NIR autofluorescence study in colonic cancer . Further, the changes of tissue microstructural scattering properties (e.g. nucleus size, refractive index, microvasculature, etc) may also attribute to the significant differences of DR spectra between normal and colonic cancer tissues . However, how the complex nature of the scattering process in pathologic tissue contributing to tissue DR spectroscopy still warrants further investigations.
To develop robust multivariate spectral diagnostic algorithms for optical diagnosis of colonic cancer, we fully utilize all the diagnostic biochemical information contained in the Mueller-matrix DR spectra acquired (Fig. 5) by implementing PLS-DA and LOSCV technique on the quantitative Mueller-matrix metrics (i.e., diattentuation, depolarization and retardance) derived using polar decomposition algorithms . Figure 6 shows the decomposed spectroscopic diattentuation ± 1 standard error (SE) (shaded area), depolarization ± 1 SE, and retardance ± 1 SE metrics of 30 paired colonic tissues. As consistent with the decomposed Mueller-Matrix images [Figs. 4(a), 4(b), 4(d), 4(e))], a significantly increased diattentuation (p<0.01) while a much reduced depolarization is observed in colonic cancer, demonstrating the potential of the Mueller-Matrix spectroscopy for colon cancer diagnosis. Remarkably, the decomposed retardance spectra [Fig. 5(b)] show a clear decrease for the colonic cancer. The changes in retardance are likely caused by the decreased collagen content in the colon cancer tissues  as the retardance effects are mainly attributed by the anisotropic orientation of collagen fibers in the concentric lamina propria and submucosa layers of the cross-section of a colon wall . The prominent differences in the decomposed retardance between normal and cancer colon tissues [Fig. 6(c)] reconfirm the capability of Mueller-Matrix NIR diffuse reflectance spectroscopy for colonic cancer detection. Further PLS-DA and LOSCV analysis implemented on the 3 derived spectroscopic polarimetric metrics (Fig. 6) shows that colon cancer was identified with an accuracy of 90.0%, 91.7%, and 80.0%, respectively, by using diattentuation, depolarization, and retardance metrics (Table 1). Remarkably, the combination of the three polarization metrics with majority voting  provides an enhanced colonic cancer detection with an accuracy of 95.0% (sensitivity of 93.3%, and specificity of 96.7%), superior to using either of the three polarization metrics alone (Table 1).
We have developed an integrated Mueller-matrix NIR imaging and point-wise Mueller-matrix spectroscopy system for optical diagnosis and characterization of colonic cancer. Point-wise Mueller-Matrix spectra can be acquired immediately under Mueller-matrix imaging guidance. Significantly increased diattentuation while significantly reduced depolarization and retardance effects were observed associated with colonic cancer. Using the combined decomposed spectroscopic polarimetric metrics (i.e., diattentuation, depolarization, and retardance), colonic cancer can be identified with a high diagnostic accuracy (~95%). This work shows that Mueller-matrix NIR imaging and point-wise Mueller-Matrix NIR diffuse reflectance spectroscopy technique may open a new avenue for enhancing optical detection and diagnosis of colonic cancer in gastrointestinal tracts.
This work was supported by the National Medical Research Council (NMRC) (Grant Numbers: CIRG/1331/2012; BnB13dec037; BnB/0012c/2014), and the Academic Research Fund (AcRF)-Tier 2 from Ministry of Education (MOE) (Grant Number: MOE2014-T2-1-010), Singapore.
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