Polarization-sensitive second harmonic generation (SHG) microscopy is an established imaging technique able to provide information related to specific molecular structures including collagen. In this investigation, polarization-sensitive SHG microscopy was used to investigate changes in the collagen ultrastructure between histopathology slides of normal and diseased human thyroid tissues including follicular nodular disease, Grave’s disease, follicular variant of papillary thyroid carcinoma, classical papillary thyroid carcinoma, insular or poorly differentiated carcinoma, and anaplastic or undifferentiated carcinoma ex vivo. The second-order nonlinear optical susceptibility tensor component ratios, χ(2)zzz′/χ(2)zxx′ and χ(2)xyz′/χ(2)zxx′, were obtained, where χ(2)zzz′/χ(2)zxx′ is a structural parameter and χ(2)xyz′/χ(2)zxx′ is a measure of the chirality of the collagen fibers. Furthermore, the degree of linear polarization (DOLP) of the SHG signal was measured. A statistically significant increase in χ(2)zzz′/χ(2)zxx′ values for all the diseased tissues except insular carcinoma and a statistically significant decrease in DOLP for all the diseased tissues were observed compared to normal thyroid. This finding indicates a higher ultrastructural disorder in diseased collagen and provides an innovative approach to discriminate between normal and diseased thyroid tissues that is complementary to standard histopathology.
Subscribe to Journal
Get full journal access for 1 year
only $36.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
American Cancer Society. Cancer facts & figures. Atlanta: American Cancer Society; 2019. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2019/cancer-facts-and-figures-2019.pdf.
Hundahl SA, Fleming ID, Fremgen AM, Menck HR. A National Cancer Data Base report on 53,856 cases of thyroid carcinoma treated in the U.S., 1985–1995. Cancer. 1998;83:2638–48.
LiVolsi VA, Asa SL. The demise of follicular carcinoma of the thyroid gland. Thyroid. 1994;4:233–6.
Schmidbauer B, Menhart K, Hellwig D, Grosse J. Differentiated thyroid cancer—treatment: state of the art. Int J Mol Sci. 2017;18:1292.
Justin EP, Seabold JE, Robinson RA, Walker WP, Gurll NJ, Hawes DR. Insular carcinoma: a distant thyroid carcinoma with associated iodine-131 localization. J Nucl Med. 1991;32:1358–63.
American Thyroid Association. Anaplastic thyroid cancer. Falls Church: American Thyroid Association; 2017. https://www.thyroid.org/wp-content/uploads/patients/brochures/anaplastic-thyroid-cancer-brochure.pdf.
Kazaure HS, Roman SA, Sosa JA. Insular thyroid cancer a population-level analysis of patient characteristics and predictors of survival. Cancer. 2012;118:3260–7.
Lam K, Lo C, Chan K, Wan K. Insular and anaplastic carcinoma of the thyroid: a 45-year comparative study at a single institution and a review of the significance of p53 and p21. Ann Surg. 2000;231:329–38.
Lloyd RV, Erickson LA, Casey MB, Lam KY, Lohse CM, Asa SL, et al. Observer variation in the diagnosis of follicular variant of papillary thyroid carcinoma. Am J Surg Pathol. 2004;28:1336–40.
Elsheikh TM, Asa SL, Chan JKC, DeLellis RA, Heffess CS, LiVolsi VA, et al. Interobserver and intraobserver variation among experts in the diagnosis of thyroid follicular lesions with borderline nuclear features of papillary carcinoma. Am J Clin Pathol. 2008;130:736–44.
Hirokawa M, Carney JA, Goellner JR, DeLellis RA, Heffess CS, Katoh R, et al. Observer variation of encapsulated follicular lesions of the thyroid gland. Am J Surg Pathol. 2002;26:1508–14.
Golaraei A, Cisek R, Krouglov S, Navab R, Niu C, Sakashita S, et al. Characterization of collagen in non-small cell lung carcinoma with second harmonic polarization microscopy. Biomed Opt Express. 2014;5:3562–7.
Gailhouste L, Grand YL, Odin C, Guyader D, Turlin B, Ezan F, et al. Fibrillar collagen scoring by second harmonic microscopy: A new tool in the assessment of liver fibrosis. J Hepatol. 2010;52:398–406.
Liu F, Chen L, Rao HY, Teng X, Ren YY, Lu YQ, et al. Automated evaluation of liver fibrosis in thioacetamide, carbon tetrachloride, and bile duct ligation rodent models using second-harmonic generation/two-photon excited fluorescence microscopy. Lab Investig. 2017;97:84–92.
Cicchi R, Kapsokalyvas D, De Giorgi V, Maio V, Van Wiechen A, Massi D, et al. Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy. J Biophoton. 2010;3:34–43.
Zhuo S, Yan J, Chen G, Shi H, Zhu X, Lu J, et al. Label-free imaging of basement membranes differentiates normal, precancerous, and cancerous colonic tissues by second-harmonic generation microscopy. PLoS ONE. 2012;7:e38655.
Adur J, Pelegati VB, de Thomaz AA, Baratti MO, Andrade LALA, Carvalho HF, et al. Second harmonic generation microscopy as a powerful diagnostic imaging modality for human ovarian cancer. J Biophotonic. 2014;7:37–48.
Burke K, Smid M, Dawes RP, Timmermans MA, Salzman P, van Deurzen CHM, et al. Using second harmonic generation to predict patient outcome in solid tumors. BMC Cancer. 2015;15:929.
Ling Y, Li C, Feng K, Palmer S, Appleton PL, Lang S, et al. Second harmonic generation (SHG) imaging of cancer heterogeneity in ultrasound guided biopsies of prostate in men suspected with prostate cancer. J Biophoton. 2017;10:911–8.
Yuting L, Li C, Zhou K, Guan G, Appleton PL, Lang S, et al. Microscale characterization of prostate biopsies tissues using optical coherence elastography and second harmonic generation imaging. Lab Investig. 2018;98:380–90.
Hristu R, Eftimie LG, Stanciu SG, Tranca DE, Paun B, Sajin M, et al. Quantitative second harmonic generation microscopy for the structural characterization of capsular collagen in thyroid neoplasms. Biomed Opt Express. 2018;9:3923–36.
Freund I, Deutsch M, Sprecher A. Connective tissue polarity. Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon. Biophys J. 1986;50:693–712.
Stoller P, Kim BM, Rubenchik AM, Reiser KM, Da Silva LB. Polarization-dependent optical second-harmonic imaging of a rat-tail tendon. J Biomed Opt. 2002;7:205–14.
Stoller P, Reiser KM, Celliers PM, Rubenchik AM. Polarization-modulated second harmonic generation in collagen. Biophys J. 2002;82:3330–42.
Stoller P, Celliers PM, Reiser KM, Rubenchik AM. Quantitative second-harmonic generation microscopy in collagen. Appl Opt. 2003;42:5209–19.
Williams RM, Zipfel WR, Webb WW. Interpreting second-harmonic generation images of collagen I fibrils. Biophys J. 2005;88:1377–86.
Erikson A, Ortegren J, Hompland T, Davies CD, Lindgren M. Quantification of the second-order nonlinear susceptibility of collagen I using a laser scanning microscope. J Biomed Opt. 2007;12:1–10.
Tiaho F, Recher G, Rouède D. Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy. Opt Express. 2007;15:12286–95.
Nadiarnykh O, Campagnola PJ. Retention of polarization signatures in SHG microscopy of scattering tissues through optical clearing. Opt Express. 2009;17:5794–806.
Gusachenko I, Latour G, Schanne-Klein M-C. Polarization-resolved second harmonic microscopy in anisotropic thick tissues. Opt. Express. 2010;18:19339–52.
Su PJ, Chen WL, Chen YF, Dong CY. Determination of collagen nanostructure from second-order susceptibility tensor analysis. Biophys J. 2011;100:2053–62.
Ambekar R, Lau T-Y, Walsh M, Bhargava R, Toussaint KC. Quantifying collagen structure in breast biopsies using second-harmonic generation imaging. Biomed Opt Express. 2012;3:2021–35.
Golaraei A, Kontenis L, Cisek R, Tokarz D, Done SJ, Wilson BC, et al. Changes of collagen ultrastructure in breast cancer tissue determined by second-harmonic generation double stokes-mueller polarimetric microscopy. Biomed Opt Express. 2016;7:4054–68.
Okoro C, Kelkar V, Sivaguru M, Emmadi R, Toussaint KC. Second-harmonic patterned polarization-analyzed reflection confocal microscopy of stromal collagen in benign and malignant breast tissues. Sci Rep. 2018;8:16243.
Campbell KR, Campagnola PJ. Assessing local stromal alterations in human ovarian cancer subtypes via second harmonic generation microscopy and analysis. J Biomed Opt. 2017;22:116008.
Campbell KR, Chaudhary R, Handel JM, Patankar MS, Campagnola PJ. Polarization-resolved second harmonic generation imaging of human ovarian cancer. J Biomed Opt. 2018;23:066501.
Birk JW, Tadros M, Moezardalan K, Nadyarnykh O, Forouhar F, Anderson J, et al. Second harmonic generation imaging distinguishes both high-grade dysplasia and cancer from normal colonic mucosa. Dig Dis Sci. 2014;59:1529–34.
Hristu R, Stanciu SG, Tranca DE, Stanciu GA. Improved quantification of collagen anisotropy with polarization-resolved second harmonic generation microscopy. J Biophoton. 2017;10:1171–9.
Tokarz D, Cisek R, Golaraei A, Krouglov S, Navab R, Niu C, et al. Tumor tissue characterization using polarization-sensitive second harmonic generation microscopy. Proc. SPIE. 2015;9531:95310C.
Tokarz D, Cisek R, Joseph A, Golaraei A, Mirsanaye K, Krouglov S, et al. Characterization of pancreatic cancer tissue using multiphoton excitation fluorescence and polarization-sensitive harmonic generation microscopy. Front Oncol. 2019;9:1–10.
Tokarz D, Cisek R, Golaraei A, Asa SL, Barzda V, Wilson BC. Ultrastructural features of collagen in thyroid carcinoma tissue observed by polarization second harmonic generation microscopy. Biomed Opt Express. 2015;6:3475–81.
Tuer AE, Krouglov S, Prent N, Cisek R, Sandkuijl D, Yasufuku K, et al. Nonlinear optical properties of type I collagen fibers studied by polarization dependent second harmonic generation microscopy. J Phys Chem B. 2011;115:12759–69.
Major A, Cisek R, Sandkuijl D, Barzda V. Femtosecond Yb:KGd(WO4)2laser with > 100 nJ of pulse energy. Laser Phys Lett. 2009;6:272–4.
Greenhalgh C, Prent N, Green C, Cisek R, Major A, Stewart B, et al. Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes. Appl Opt. 2007;46:1852–9.
Carriles R, Schafer DN, Sheetz KE, Field JJJ, Cisek R, Barzda V, et al. Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy. Rev Sci Instrum. 2009;80:10364–71.
Tuer AE, Akens MK, Krouglov S, Sandkuijl D, Wilson BC, Whyne CM, et al. Hierarchical model of fibrillar collagen organization for interpreting the second-order susceptibility tensors in biological tissue. Biophys J. 2012;103:2093–105.
Golaraei A, Mirsanaye K, Ro Y, Krouglov S, Akens MK, Wilson BC, et al. Collagen chirality and three-dimensional orientation studied with polarimetric second-harmonic generation microscopy. J Biophoton. 2019;12:e201800241.
Golaraei A, Kontenis L, Mirsanaye K, Krouglov S, Akens MK, Wilson BC, et al. Complex susceptibilities and chiroptical effects of collagen measured with polarimetric second-harmonic generation microscopy. Sci Rep. 2019;9:124881–12.
Asa SL. Pathology survival guides, series 1, number 4: survival guide to endocrine pathology. Arlington: Innovative Science Press; 2020.
This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN-2017-06923 and DGDND-2017-00099) and the Canadian Institutes of Health Research (CIHR) through a Collaborative Health Research Project (CHRP) grant (CPG-134752 and CHRPJ 462842-14). The authors thank the staff of the Advanced Optical Microscopy Facility at University Health Network for assistance with whole-slide scanning.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a real or potential conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Tokarz, D., Cisek, R., Joseph, A. et al. Characterization of pathological thyroid tissue using polarization-sensitive second harmonic generation microscopy. Lab Invest (2020). https://doi.org/10.1038/s41374-020-0475-7