Saratov Fall Meeting 2016 Wavelength dependence of refractive index of human colon tissues: comparison between healthy mucosa and tumor polyps Sónia Carvalho1, Nuno Gueiral2, Elisabete Nogueira2, Rui Henrique1, Luís Oliveira2, Valery V. Tuchin3,4,5 1Serviço de Anatomia Patológica – IPO Porto, Rua Dr. António Bernardino de Almeida S/N, 4200-072 Porto, Portugal 3Research-Education Institute of Optics and Biophotonics, Saratov National Research State University, 83 Astrakhanskaya str., Saratov 410012, Russia 4Laboratory of Laser Diagnostics of Technical and Living Systems, Precision Mechanics and Control Institute of the Russian Academy of Sciences, 24 Rabochaya, Saratov 410028, Russia 2CIETI/ISEP – Physics Department, Rua Dr. António Bernardino de Almeida Nº 431, 4249-015 Porto, Portugal 5Interdisciplinary Laboratory of Biophotonics, National Research Tomsk State University, 36 Lenin’s av., Tomsk 634050, Russia

Introduction and motivation The third most common cancer and the fourth to cause death worldwide is colorectal cancer. Approximately 1.2 million new cases are diagnosed every year worldwide and 50% of this value is converted into patient death (Brenner et al, 2014) Current technology implies colon cross-section biopsy to establish a reliable diagnosis. Noninvasive optical methods are desired to turn the diagnosis/treatment procedures easier and less aggressive to the patient The colon tube consists of several layers from the inside to the outside Source: https://www.ouhsc.edu/histology/Glass slides/56_01.jpg

Colorectal cancer polyps start developing in the innermost colon tube layer – the mucosa (Subramaniam et al, 2016) Source: http://en.paperblog.com/prolonged-sitting-associated-with-colon-polyps-and-cancer-1127955/ The development of the this type of cancer is accompanied with polyp growth into the deeper layers of the colon (sub-mucosa and muscle) (Pierangelo et al, 2011) Since the colorectal cancer polyps start developing in the mucosa, endoscopic instrumentation can be inserted into the colon to perform early cancer detection

Early this year, a research group has demonstrated that colon mucosa and dysplastic colon tissues present different refractive index (RI) values at 964 nm (Giannios et al, 2016) Since the RI is an optical property that can be directly measured, The discrimination of this parameter between healthy and pathological mucosa can be used for diagnostic purposes Considering that optical diagnostic and treatment procedures can operate at different wavelengths, we have studied the wavelength dependence of the RI of colon healthy and pathological mucosa for visible and NIR ranges The results in this comparative study allow the discrimination between tumor and healthy mucosa at different wavelengths through the measurement of their RI

Materials and methods Tissue samples Gender Age - range 3 2 52 - 82 Colon tissue samples were surgically removed from a population of 5 patients within a 3 month period Healthy mucosa and cancer polyp samples were separated and preserved frozen at ‒80ºC for a period of 12-24 hrs. A thin slice was made on the top surface of samples to make them flat, so they could be used in measurements with prism A cryostat was used to prepare samples to use in measurements with Abbe refractometer (λ=589 nm). These samples had 0.4 mm thickness Before studies, samples were kept in saline for 10 min to mimic natural hydration

Measurement procedure The RI measuring procedure adopted in this study was the internal reflection method with a dispersion prism (Ye et al, 2011) (Deng et al, 2016) (Ding et al, 2006): The sample tissue was placed in contact to the base of the prism; RI measuring setup An incident beam (from a laser) was used to illuminate the setup through a lateral side of the prism; The reflected beam was detected by a photocell connected to a voltmeter to measure the potential difference; This procedure was repeated for several angles of incidence; Reflectance at the prism/tissue interface was calculated for each angle as: 𝑹 𝜽 = 𝑽 𝜽 − 𝑽 𝒏𝒐𝒊𝒔𝒆 𝑽 𝒍𝒂𝒔𝒆𝒓 − 𝑽 𝒏𝒐𝒊𝒔𝒆 Background potential Measured directly from laser

A representation of the reflectance at the prism/tissue interface as a function of the incident angle was created, showing an increase from a lower to a top value; The derivative of the previous curve is calculated showing that a peak is observed at a particular angle in the increasing reflectance of the first curve. This angle is the critical angle of reflection for a particular laser between the prism and the tissue sample; Once the critical angle (c) is determined for a particular laser, it is used to calculate the correspondent RI of the tissue: 𝒏 𝒓 = 𝒏 𝟏 ×𝒔𝒊𝒏 𝜷−𝒂𝒓𝒄𝒔𝒊𝒏 𝟏 𝒏 𝟏 ×𝒔𝒊𝒏 𝜶 𝒄  Represents the internal angle of the prism (=60°) RI of the prism at the same wavelength of the laser All this procedure was repeated three times for healthy and pathological mucosa tissues using various lasers with the following wavelengths: 532.5 nm, 670 nm, 783.2 nm, 822.7 nm and 850.7 nm. In addiction to these measurements, the RI of healthy and pathological mucosa were also measured with the Abbe refractometer (λ=589 nm). Three samples each (healthy and pathological) were prepared with 0.4 mm thickness for these measurements

Experimental results and calculations Reflectance curves at prism/tissue interface – some examples Several reflectance curves were calculated for the various studies performed with the two types of tissues and various lasers. We present here two cases (3 healthy and 3 pathological mucosa samples) for a particular laser (λ= 783.2 nm): Figures above show that in both cases reflectance increases with the incident angle. On the other hand such increase is slower in the case of the pathological mucosa.

Critical angle determination Calculation of RI(λ) for each laser was made using the critical angles observed in the 1st derivative of the reflectance curves for each case. The correspondent 1st derivative curves for the previous graphs are presented: Curves like above were fitted with a smooth spline using CFTOOL in MATLAB TM to estimate the critical angle with precision. The critical angles of reflectance correspond to the peaks in previous graphs.

Refractive index calculation The critical incident angle at the prism/sample interface (θc) has a correspondent incidence angle at the air/prism interface - 𝜶c that can be measured. These values were estimated from curves like the ones previously presented and used in following equation to calculate RI(λ) of the samples 𝒏 𝒓 = 𝒏 𝟏 ×𝒔𝒊𝒏 𝜷−𝒂𝒓𝒄𝒔𝒊𝒏 𝟏 𝒏 𝟏 ×𝒔𝒊𝒏 𝜶 𝒄 θc Since we have performed three sets of measurements for each tissue/laser, we have calculated the mean and SD values for the RI values of the healthy and pathological mucosa for the various wavelengths of the lasers used in the measurements. Similarly, mean and SD values were also calculated for the RI of both samples at the wavelength of the Abbe refractometer.

All the RI values, mean for each wavelength and correspondent SD values are presented in the following table: Mucosa 532.5 nm 589 nm 670 nm 783.2 nm 822.7 nm 850.7 nm   Tumor Sample 1 1.3482 1.3387 1.3303 1.3310 1.3426 1.3418 1.3573 1.3518 1.3462 1.3337 1.3516 1.3754 Sample 2 1.3437 1.3383 1.3341 1.3290 1.3404 1.3423 1.3572 1.3523 1.3455 1.3332 1.3514 1.3762 Sampl3 1.3364 1.3379 1.3306 1.3399 1.3419 Sample 3 1.3529 1.3471 1.3343 Mean 1.3427 1.3336 1.3358 1.3409 1.3420 1.3463 1.3515 1.3759 SD 0.0600 0.0004 0.0031   0.0011 0.0014  0.0002   0.0006 0.0006  0.0008  0.0001  0.0005 The mean and standard deviation bars for healthy mucosa are presented in next figure along with the mucosa dispersion curve calculated with (Giannios et al, 2016) : 𝒏 𝑴𝑼𝑪𝑶𝑺𝑨 =1.3217+ 0.00994 𝝀 𝟐 − 0.00089 𝝀 𝟒

As we could see from previous figure, our experimental results are a little lower than the theoretical curve presented in literature (Giannios et al, 2016). Such difference might be related to the geographical origin of samples and possibly due to eventual small air bubbles at the prism/tissue interface (Deng et al, 2016) The previous graph also shows that for the two longest wavelengths we used, RI does not fit the Cauchy dispersion curve. Tumor tissue presents same tendency and with higher increase: Such non-monotonic behavior at longer wavelengths can be explained by the presence of lipids in tissues that have absorption bands in this range (Van Veen et al, 2005). For these wavelengths the absorption and scattering coefficients have similar values and consequently RI increases.

Conclusions and future perspectives We have estimated the wavelength dependence of the RI for healthy and pathological mucosa Such wavelength dependence for the healthy mucosa is according to literature data, but probably due to geographical origin of samples and possibly bad coupling of tissue samples to prism surface, our results are a little lower that the theoretical curve created from Greek data Pathological and healthy mucosa present similar wavelength dependence, but data from pathological samples presents higher RI values for visible and NIR wavelength range By measuring at 822 and 850 nm, we have obtained non monotonic wavelength dependence for the RI of mucosa tissues. Such behavior might be related to the presence of lipids in the samples we used. Lipids have strong absorption bands in this range of spectra and the similar levels of scattering and absorption coefficients originates an increase in the RI

In the near future we will repeat these measurements and perform others for smaller and longer wavelengths to obtain a statistically reliable wavelength dependence for the RI of mucosa tissues (healthy and pathological) in a wider spectral range Combining such RI data with optical measurements we plan to estimate the optical properties of mucosa tissues to discriminate between healthy and pathological Once these estimations are completed, we can perform similar comparative studies between healthy and pathological tissues from different anatomical areas of the human body

Acknowledgements The authors are thankful for the Portuguese Research grant FCT-UID/EQU/00305/2013 VVT was supported by the Russian Presidential grant NSh-7898.2016.2, the Russian Governmental grant 14.Z50.31.0004, and The National Research Tomsk State University Academic D.I. Mendeleev Fund Program The authors appreciate the availability of instrumental and technical resources from I3S-Porto and the help of Cláudia Machado in preparing the tissue samples

References Brenner H., Kloor M., Pox C. P. 2014, Colorectal cancer, The Lancet, vol. 383, pp. 1490-1502. https://www.ouhsc.edu/histology/Glass slides/56_01.jpg. Subramaniam R., Mizogushi A. Mizogushi E. 2016, Mechanistic roles of epithelial and immune cell signaling during the development of colitis-associated cancer, Cancer Research Frontiers, vol. 2, pp. 1-21. http://en.paperblog.com/prolonged-sitting-associated-with-colon-polyps-and-cancer-1127955/ Pierangelo A., Benali A., Antonelli M. R., Novikova T., Validire P., Gayet B., De Martino A. 2011, Ex-vivo characterization of human colon cancer by Mueller polarimetric imaging, Optics Express, vol. 19, pp. 1582-1593. Giannios P., Koutsoumpos S., Toutouzas K. G., Matiatou M., Zografos G. C., Moutzaouris K. 2016, Complex refractive index of normal and malignant human colorectal tissue in the visible and near-infrared, J. of Biophotonics – online publication. Ye Q., Wang J., Deng Z.-C., Zhou W.-Y., Zhang C.-P., Tian J.-G. 2011, Measurement of the complex refractive index of tissue-mimicking phantoms and biotissue by extended differential total reflection method, J. Biomed. Opt., vol. 16, pp. 097001-1-5. Deng Z., Wang J. Ye Q., Sun T., Zhou W., Mei J., Zhang C., Tian J. 2016, Determination of continuous complex refractive dispersion of biotissue based on internal reflection, J. Biomed. Opt., vol. 21, pp. 015003. Ding H., Lu J. Q., Wooden W. A., Kragel P. J., Hu X.-H. 2006, Refractive indices of human skin tissues at eight wavelengths and estimated dispersion relations between 300 and 1600 nm, Phys. Med. Biol., vol.51, pp.1479-1489. van Veen R. L. P., Sterenborg H. J. C. M., Pifferi A., Torricelli A., Chikoidze E., Cubeddu R. 2005, Determination of visible near-IR absorption coefficients of mammalian fat using time- and spatially resolved diffuse reflectance and transmission spectroscopy, J. Biomed. Opt., vol. 10, pp. 054004-1-6.

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