In this study, we utilize the properties of polarized light for the analysis of paraffin-embedded breast cancer samples. We perform the measurements of the full Stokes vector of back-reflected radiation and calculate the degree of polarization as a diagnostic criterion for the separation of healthy and cancer sample sections. We show that circularly polarized light scattered within the breast sample is sensitive to the presence of cancer cells. The degree of the polarization of the reected light was found to be the most sensitive parameter for the reliable differentiation of tissue

Anin, Dmytro

Borovkova, Mariia

Bykov, Alexander

Dremin, Viktor

Meglinski, Igor

Näpänkangas, Juha

Sieryi, Oleksii

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PROCEEDINGS OF SPIESPIEDigitalLibrary.org/conference-proceedings-of-spieImaging of early stage breast cancerwith circularly polarized lightDremin, Viktor, Anin, Dmytro, Sieryi, Oleksii, Borovkova,Mariia, Näpänkangas, Juha, et al.Viktor Dremin, Dmytro Anin, Oleksii Sieryi, Mariia Borovkova, JuhaNäpänkangas, Igor Meglinski, Alexander Bykov, "Imaging of early stagebreast cancer with circularly polarized light," Proc. SPIE 11363, Tissue Opticsand Photonics, 1136304 (7 April 2020); doi: 10.1117/12.2554166Event: SPIE Photonics Europe, 2020, Online OnlyDownloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 16 Dec 2020  Terms of Use: https://www.spiedigitallibrary.org/terms-of-useImaging of early stage breast cancer withcircularly polarized lightViktor Dremina,b, Dmytro Anina, Oleksii Sieryia, Mariia Borovkovaa,Juha Näpänkangasc, Igor Meglinskia,d,e, and Alexander BykovaaOptoelectronics and Measurement Techniques Unit, University of Oulu, Oulu, FinlandbResearch & Development Center of Biomedical Photonics, Orel State University, Orel, RussiacOulu University Hospital, University of Oulu, Oulu, FinlanddSchool of Engineering & Applied Science, Aston University, Birmingham, UKeSchool of Life & Health Sciences, Aston University, Birmingham, UKABSTRACTIn this study, we utilize the properties of polarized light for the analysis of paraffin-embedded breast cancersamples. We perform the measurements of the full Stokes vector of back-reflected radiation and calculate thedegree of polarization as a diagnostic criterion for the separation of healthy and cancer sample sections. Weshow that circularly polarized light scattered within the breast sample is sensitive to the presence of cancer cells.The degree of the polarization of the reflected light was found to be the most sensitive parameter for the reliabledifferentiation of tissue.Keywords: breast cancer, Stokes polarimetry, polarimetric imaging, degree of polarization1. INTRODUCTIONBreast carcinoma is the most widespread cancer among women with more than 400 000 cases diagnosed each yearacross EU countries.1 For successful treatment, the early detection of cancer is extremely important. Millions ofwomen are surviving and thriving with breast cancer when found and treated early. Currently, the only way andgold standard to set definite diagnostics of cancer is histology. However, despite the best laboratory practices,the rate of conclusive diagnosis by histological analysis for a range of cancers is only 65-75%.2 Typically, thebiopsy is taken at the time of surgery operation with a high demand for immediate qualitative pathologicalreview. The development of an efficient diagnostic modality may revolutionize the current practice of cancerdiagnosis and treatment. The ability to recognize the suspect tissue in a short time without the interruption ofthe operation is able to increase the effectiveness of the surgery of the cancerous tumors.Many imaging studies use the polarized light as a supplementary tool e.g. to reduce the specular reflectionfrom the surface of the measured object.3 However, recent research4–12 demonstrates the potential of bulkpolarimetry as a standalone modality for the biotissue diagnosis. In this study, we suggest using the propertiesof polarized light for the analysis of formalin-fixed paraffin-embedded (FFPE) breast cancer samples. We proposemeasurements of the Stokes vector and the calculation of the degree of polarization (DOP) as a diagnostic criterionfor the separation of healthy and cancer sample sections. The standard routine procedure for the histologicalanalysis uses a number of sample preparation steps, which include fixation of the sample in formalin, ethanol,etc.; dehydration and embedding into the paraffin wax or celloidin; sectioning with a microtome; mounting onglass slides, staining by hematoxylin, eosin, etc.; and finally microscopy investigation.13 Thus, the conventionalhistological analysis is rather laborious, time-consuming and strongly relies on the skills and qualification of thepathologist performing the examination. However, the proposed approach allows us to avoid several steps in theconventional histology procedure and perform the tissue sample analysis right after the embedding step.Send correspondence to Viktor DreminE-mail: viktor.dremin@oulu.fi,Telephone: +358 41 470 6340Invited PaperTissue Optics and Photonics, edited by Valery V. Tuchin, Walter C. P. M. Blondel, Zeev Zalevsky, Proc. of SPIE Vol. 11363, 1136304 · © 2020 SPIE CCC code: 0277-786X/20/$21 · doi: 10.1117/12.2554166Proc. of SPIE Vol. 11363  1136304-1Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 16 Dec 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-useFigure 1. Overview of FFPE breast cancer samples (left side) with ROI showing the enlarged area scanned withmicroscope (right side).As you can see from Fig. 1, standard microscopic examination of the paraffin-embedded tissue sample doesnot provide any useful diagnostic information due to the low contrast between tissue constituents in the un-stained sample. In the same time, the involvement of the polarization properties sensitive to the changes inthe microstructure of the sample can solve this problem as well as provide quantitative information about theanalyzed sample. In addition, our approach will reduce both the number of sample processing steps and the timeneeded for the analysis.2. MATERIAL AND METHODSDue to the high sensitivity of polarized light to the variations of scattering in the analyzed sample, the spatiallyresolved polarized light spectroscopy has a great potential to be used for screening of malformations in the bio-logical tissues.14 We have developed an experimental polarization-based imaging system for scanning biologicalsamples. In our setup, circularly polarized light was used due to its unique ability to preserve the polarizationproperties during larger number of scattering events than the linearly polarized light.15,16 The measurementsystem contains both illumination and detection channels (see Fig. 2).Figure 2. Scheme of the experimental setup containing the illumination arm (right side) and measurement arm (leftside).The radiation of supercontinuum fiber laser (Leukos Ltd., France) was used in the illumination channel. Thespecific probing wavelength was selected with the high-speed acousto-optic tunable filter (Leukos Ltd., France)from the range of 400–650 nm. The linearly polarized light from the output of the filter was then altered by aProc. of SPIE Vol. 11363  1136304-2Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 16 Dec 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-usequarter-wave plate into the state of right-hand circular polarization and focused onto the sample at 55◦ angle.The state of polarization of the light diffusely reflected from the sample was collected at 30◦ angle at a variabledistance away from the point of incidence with the 20× objective lens and analyzed by the Stokes polarimetersystem (Thorlabs Ltd., USA). Spatial scanning of the samples was performed with the XY translation stageproviding the resolution of 5 µm.The multiple measurements have been performed on a FFPE blocks of early stage breast cancer samples(ductal carcinoma in situ, DCIS) and compared to the measurements from developed cancer samples (invasiveductal carcinoma, IDC). Breast cancer samples and their histology were provided by the Northern FinlandBiobank Borealis (Oulu, Finland).3. RESULTS AND DISCUSSIONWe show that circularly polarized light scattered within the breast sample is sensitive to the presence of cancercells and provides the information close to that obtained from the standard histological examination (see Fig. 3).However, no staining and no slicing is required in our case.Figure 3. Histological image of DCIS (a) and 2D spatial distributions of the degree of polarization of the reflected lightmeasured at 450 nm (b). Histological image of IDC and benign fibrosis (c) and 2D spatial distributions of the degree ofpolarization (d).The highest contrast between cancerous and normal regions was observed for the probing wavelength of 450nm. The DOP of the reflected light was found to be the most sensitive parameter indicating the malignantchanges in the considered samples. DOP shows how much of light is polarized within the whole light that isdetected. The average DOP values for intact adipose tissue were 95%, for DCIS – 70-80%, the presence of IDCreduced the DOP value to the level of 60-70%. The proposed methodology demonstrates a great potential fordeveloping a new diagnostic protocol for routine clinical practice.And finally, a statistically significant differences confirmed by Kruskal-Wallis test with the post-hoc analysis(p < 0.01) exists between four types of tissue in the DOP parameter. The observed differences may be related toan increase in the average size of the nuclei in a more developed type of cancer. This leads to higher anisotropyand an increase in successive forward scattering events, which reduces the DOP.4. CONCLUSIONA circular polarization imaging system for obtaining histological images was developed. We have shown thancircularly polarised light scattered within breast samples is sensitive to the presence of cancer cells and thehighest perceptual contrast between cancerous and normal regions was observed for the probing wavelength of450 nm. The DOP of the reflected light was found to be the most sensitive parameter indicating the malignantchanges in the considered samples. The proposed methodology demonstrates a great potential for developing anew diagnostic protocol for routine clinical practice.Proc. of SPIE Vol. 11363  1136304-3Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 16 Dec 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-useACKNOWLEDGMENTSAuthors acknowledge the support of the Academy of Finland (grants: 314369 and 325097), the ATTRACTproject funded by the EU under Grant Agreement 777222. This study was also supported by the Russian ScienceFoundation under projects No. 19-79-00082 (experimental data collection). Authors acknowledge NorthernFinland Biobank Borealis, Oulu, Finland (www.ppshp.fi/biopankki) for providing the breast cancer samples.REFERENCES[1] [Health at a Glance: Europe 2018: State of Health in the EU Cycle ], OECD Publishing, Paris (2018).[2] Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., and Jemal, A., “Global cancer statistics2018: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries,” CA: ACancer Journal for Clinicians 68(6), 394–424 (2018).[3] Zherebtsov, E., Dremin, V., Popov, A., Doronin, A., Kurakina, D., Kirillin, M., Meglinski, I., and Bykov,A., “Hyperspectral imaging of human skin aided by artificial neural networks,” Biomed. Opt. Express 10(7),3545–3559 (2019).[4] Novikova, T., Meglinski, I., Ramella-Roman, J., and Tuchin, V., “Polarized light for biomedical applica-tions,” J. Biomed. Opt. 21(7), 071001 (2016).[5] Ushenko, V., Sdobnov, A., Syvokorovskaya, A., Dubolazov, A., Vanchulyak, O., Ushenko, A., Ushenko,Y., Gorsky, M., Sidor, M., Bykov, A., and Meglinski, I., “3D Mueller-matrix diffusive tomography ofpolycrystalline blood films for cancer diagnosis,” Photonics. 5, 54 (2018).[6] Ushenko, A., Sdobnov, A., Dubolazov, A., Gritsuk, M., Ushenko, Y., Bykov, A., and Meglinski, I., “Stokes-correlometry analysis of biological tissues with polycrystalline structure,” IEEE J. Sel. Top. QuantumElectron. 25(1), 7101612 (2019).[7] Borovkova, M., Ushenko, V. A., Dubolazov, A. V., Vanchulyak, O. Y., Ushenko, O. G., Bykov, A. V.,and Meglinski, I., “Mueller-matrix-based polarization imaging and quantitative assessment of opticallyanisotropic polycrystalline networks,” PLOS One 14(5), e0214494 (2019).[8] Borovkova, M., Peyvasteh, M., Ushenko, Y. O., Dubolazov, O. V., Ushenko, V. O., Bykov, A. V., Novikova,T. P., and Meglinski, I., “Complementary analysis of Muller-matrix images of optically anisotropic highlyscattering biological tissues,” J. Eur. Opt. Soc.-Rapid. 14, 20 (2018).[9] Ushenko, V. A., Sdobnov, A. Y., Mishalov, W. D., Dubolazov, A. V., Olar, O. V., Bachinskyi, V. T.,Ushenko, A. G., Ushenko, Y. A., Wanchuliak, O. Y., and Meglinski, I., “Biomedical applications of jones-matrix tomography to polycrystalline films of biological fluids,” J. Innov. Opt. Heal. Sci. 12(6), 1950017(2019).[10] Ushenko, V. A., Dubolazov, A. V., Pidkamin, L. Y., Sakchnovsky, M. Y., Bodnar, A. B., Ushenko, Y. A.,Ushenko, A. G., Bykov, A., and Meglinski, I., “Mapping of polycrystalline films of biological fluids utilizingthe Jones-matrix formalism,” Laser Phys. 28(2), 025602 (2018).[11] Das, N. K., Dey, R., Chakraborty, S., Panigrahi, P. K., Meglinski, I., and Ghosh, N., “Quantitative assess-ment of submicron scale anisotropy in tissue multifractality by scattering Mueller matrix in the frameworkof born approximation,” Opt. Commun. 413, 172–178 (2018).[12] Das, N. K., Dey, R., Chakraborty, S., Panigrahi, P. K., Meglinski, I., and Ghosh, N., “Submicron scaletissue multifractal anisotropy in polarized laser light scattering,” Laser Phys. Lett. 15(3), 035601 (2018).[13] Nozdrin, V., Belousova, T., Piavchenko, G., and Volkov, Y., [A concise guide to histology ], Retinoids,Moscow (2019).[14] Kunnen, B., Macdonald, C., Doronin, A., Jacques, S., Eccles, M., and Meglinski, I., “Application of circu-larly polarized light for non-invasive diagnosis of cancerous tissues and turbid tissue-like scattering media,”J. Biophotonics. 8, 317–323 (2015).[15] Xu, M. and Alfano, R., “Circular polarization memory of light,” Phys. Rev. E 72(6), 065601 (2005).[16] MacKintosh, F., Zhu, J., Pine, D., and Weitz, D., “Polarization memory of multiply scattered light,” Phys.Rev. B 40(13), 9342 (1989).Proc. of SPIE Vol. 11363  1136304-4Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 16 Dec 2020Terms of Use: https://www.spiedigitallibrary.org/terms-of-use

Imaging of early stage breast cancer with circularly polarized light

Abstract

In this study, we utilize the properties of polarized light for the analysis of paraffin-embedded breast cancer samples. We perform the measurements of the full Stokes vector of back-reflected radiation and calculate the degree of polarization as a diagnostic criterion for the separation of healthy and cancer sample sections. We show that circularly polarized light scattered within the breast sample is sensitive to the presence of cancer cells. The degree of the polarization of the reflected light was found to be the most sensitive parameter for the reliable differentiation of tissue. We show that circularly polarized light scattered within the breast sample is sensitive to the presence of cancer cells. The highest contrast between cancerous and normal regions was observed for the probing wavelength of 450 nm. The degree of the polarization of the reflected light was found to be the most sensitive parameter. Mapping the Stokes vectors of backscattered light on a Poincaré sphere helps to highlight the changes of polarization state

Imaging of early stage breast cancer with circularly polarized light

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