Ultrastructural features of collagen in thyroid carcinoma tissue observed by polarization second harmonic generation microscopy

: Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.


Introduction
Thyroid cancers are the most common malignancy of the endocrine organs [1] and the incidence rate of differentiated thyroid cancers has increased significantly over the past decade [2]. Papillary thyroid carcinoma is the most prevalent form, accounting for 85-90% of all thyroid cancer cases, while follicular thyroid carcinoma accounts for <10% [3,4]. Most papillary and follicular thyroid cancers can be treated successfully when diagnosed early. Diagnosis of thyroid cancer is confirmed by visualizing stained thyroid tissue with brightfield microscopy. However, morphological interpretation of tissue by standard histopathology can be controversial with significant inter-observer variability [5,6], so that novel automated technologies with high spatial resolution and improved disease specificity are needed to aid pathologists in identifying cancer on biopsy or surgical resection sections.
Extracellular matrix (ECM) development is often deregulated and becomes disorganized during cancer initiation and progression. In principle then, cancer diagnosis can be improved by developing a technique that identifies and quantifies structural details in the ECM. Since collagen is the most abundant protein in the ECM and is known to have strong second harmonic generation (SHG) signals without staining, its content and spatial micro-distribution can be visualized by SHG microscopy [7]. Further, by applying polarization-sensitive measurements, the second-order nonlinear optical susceptibility tensor component ratio, χ (2) ZZZ '/χ (2) ZXX ', and the degree of linear polarization, DOLP, can be measured. These metrics provide additional information on the collagen ultrastructural characteristics that is complementary to the concentration and spatial distribution measures. Polarization-sensitive SHG microscopy has been previously applied to determine differences in normal and malignant breast tissue [8], models of ovarian cancer [9], and non-small cell lung carcinoma [7]. In this study, we use polarization-in, polarization-out (PIPO) SHG microscopy to investigate classical papillary thyroid carcinoma (cPTC) and follicular variant papillary thyroid carcinoma (FVPTC).

Polarization-sensitive SHG measurements with a nonlinear optical microscope
For imaging, an in-house system consisting of a ytterbium-ion-doped potassium gadolinium tungstate (Yb:KGW) crystal-based laser oscillator operating at 1028 nm with 14.3 MHz repetition rate and ~450 fs pulse duration [10] was coupled to an in-house nonlinear optical microscope. Excitation was achieved with a 0.75 numerical aperture (NA) objective lens (Carl Zeiss, Thornwood, NY, USA), while emission occurred in the transmission geometry with a 0.85 NA objective (fabricated in-house). The high NA lenses are expected to produce ~5% overestimation of χ (2) ZZZ '/χ (2) ZXX ' [11]. We assume that the induced axial polarization component is negligible. The PIPO SHG measurement consisted of recording 81 images at different combinations of orientation angles by rotating a half-wave plate placed directly before the excitation objective lens, and a polarizer (analyzer) located after the collection objective before the detector, resulting in excitation and SHG polarization orientation ranges of −90° to 90° [12]. To ensure that the input beam is linearly polarized, a linear polarizer was placed directly before the half-wave plate, and there were no optical elements after the linear polarizer, such as dichroic or dielectric mirrors, that could affect the polarization state.

Polarization-sensitive SHG image analysis
A laboratory Cartesian coordinate system, XYZ, is defined such that XZ is the image plane and the laser propagates along the Y-direction. The average orientation of collagen fibers in a pixel is defined by modified spherical angles, α and δ, where α is the angle between the axis of the collagen fibers and their projection onto the image plane, while δ is the angle between the laboratory frame Z axis and that projection. The collagen fibers are associated with their own Cartesian coordinates, xyz, where the z-axis is oriented along the collagen fiber axis. The measured second-order nonlinear optical susceptibility tensor component ratio, χ (2) ZZZ '/χ (2) ZXX ', for a sample voxel that contains cylindrical fibers of collagen [13,14] with an intrinsic second-order nonlinear optical susceptibility tensor component ratio, χ (2) zzz /χ (2) zxx , oriented at angles α and δ is given by [12]: The χ (2) ZZZ '/χ (2) ZXX ' for each pixel of the SHG image is fitted to the PIPO SHG measurements, where the SHG intensity, I 2ω , is expressed as a function of the orientation of linear polarization of the laser, θ'=θ+δ, and the analyzer orientation, φ'=φ+δ. A is the fitted noise and B is the fitted amplitude [12]. Thus: Equation (2) is valid for the case of cylindrical symmetry and birefringence can be neglected due to the minimal tissue thickness. The polarization dependence of incoherent SHG (I incoh ) can be described by Eq. (2) at 4 orthogonal values of the fiber orientation δ, yielding: From the PIPO SHG meausrements, the DOLP of the outgoing SHG was obtained from: where s 0 , s 1 and s 2 are Stokes parameters describing the SHG polarization: specifically, s 0 = I 0 + I 90 , s 1 = I 0 -I 90 , and s 2 = I 45 -I -45 , where I a is the SHG intensity at the analyzer angle a. According to double Stokes Mueller polarimetry [15], a measurement of the DOLP depends on the angle between the incident polarization of the laser and the measured collagen fiber angle (δ). When the laser polarization orientation is 90° from the collagen axis, SHG intensity is reduced and noise dominates the signal, rendering the DOLP unreliable. Nevertheless, meaningful data were obtained by averaging 16 independent DOLP measurements, 2 at each orientation of the incident laser polarization (0°, 22.5°, 45°, 67.5°, 90°, 112.5°, 135°, 157.5°).

Sample preparation
Histological sections of tumor and non-tumorous thyroid tissue were obtained according to an institutionally-approved protocol (University Health Network, Toronto). Thin (5 μm) tissue sections were cut from formalin-fixed, paraffin-embedded tissue blocks, mounted on glass slides and stained with hematoxylin and eosin (H&E). The whole sections were imaged with a bright-field microscope scanner (Aperio Whole Slide Scanner: Leica Biosystems, Germany). Assessment of both the tissue architecture and cytology of tumor cells was performed by an expert pathologist (S.L.A.) for identification of tumor and non-tumor regions of interest on the bright-field images. In H&E stained tissues, collagen gives rise to strong SHG signals [16], so that PIPO SHG scans were subsequently performed on 110 × 110 μm regions. As a control and to correct for possible photobleaching, the sample was periodically re-scanned at the initial polarizer and analyzer settings. In each sample slide, at least 2 regions of interest were imaged. In total, 16 areas in 5 patients with non-tumorous thyroid, 48 areas from 10 patients with cPTC tumors and 58 areas from 17 patients with FVPTC tumors were imaged. Two-tailed t-tests were conducted considering the number of patients imaged.
The added term, Ccos 2 (θ'-φ'), allowed for a better fit to the data, as shown in Fig. 2(e), 2(f). All the data were then re-fitted to Eq. (5) and a new metric ψ was devised to quantify the percentage of fitted pixels per image that required a significant isotropic term: where N and N' represent the number of pixels in each area that had good fit with Eqs. (2) and (5), respectively, determined by a goodness of fit linear regression parameter, R 2 >0.8.
Average ψ values in Table 1 verify the observation that FVPTC and cPTC tissues contain more elongated PIPO profiles than normal tissue (p<0.01 and <0.05, respectively). ψ shows the percentage of the affected collagenous area that contains partly disordered collagen ultrastructure. ZZZ '/χ (2) ZXX ' values were calculated for each pixel from Eq. (2), and are displayed as color-coded maps in panels (c, i, o), where blue indicates a χ (2) ZZZ '/χ (2) ZXX ' of 1 and red indicates 4. The frequency histograms of the χ (2) ZZZ '/χ (2) ZXX ' values for all the pixels are plotted in panels (d, j, p). From PIPO SHG microscopy data, DOLP values were also extracted for each pixel and are displayed as colorcoded maps in panels (e, k, q), where blue indicates a DOLP of 0 and red indicates 1. Frequency histograms of the DOLP values for all the pixels are plotted in panels (f, l, r).
The isotropic SHG contribution is reminiscent of the PIPO THG pattern produced by isotropic media [18]. Such media cannot produce coherent SHG, so that the observed contribution is attributed to incoherent hyper-Rayleigh scattered SHG from disordered collagen fibrils and small collagen fibril segments within the voxel volume. The DOLP was also calculated for each pixel to reflect the isotropic SHG contribution to the polarization plots. The SHG signals from non-tumorous thyroid tissue and tumors differed significantly in their DOLP values, indicating that tumor tissue has a greater degree of ultrastructural collagen disorder. This conclusion is somewhat similar to a previous study of the DOLP of SHG from collagen type I gels formed from bovine tendon [19]. Generally, care must be taken in interpreting DOLP data, since the circular polarization component can alter the DOLP due to SHG radiation propagation through a birefringent sample. However, since the samples used were optically thin (5 μm), the influence of birefringence and scattering due to SHG propagation through the tissue can be considered to be negligible.
In conclusion, the decrease in the DOLP values of cancerous thyroid tissue indicates that collagen becomes increasingly disordered in diseased states. The increase in χ (2) ZZZ '/χ (2) ZXX ' also indicates that the collagen structure becomes increasingly disordered. One caveat in these findings is that to date the analysis has not included samples from patients with Graves' disease in which the thyroid develops papillae or nodular hyperplasia where stromal changes occur. This will be necessary to validate the use of PIPO SHG microscopy to complement standard histopathology to improve the accuracy of thyroid cancer diagnosis and staging. There are also recent reports of implementing various nonlinear optical technqiues in vivo, either endoscopically (e.g. coherent anti-Stokes Raman, as in ref. [20]) or intraoperatively to localize tumor margins (e.g. stimulated Raman, as in ref. [21]).