Wavelength-Dependent Second Harmonic Generation Circular Dichroism for Differentiation of Col I and Col III Isoforms in Stromal Models of Ovarian Cancer Based on Intrinsic Chirality Differences

Abstract

Extensive remodeling of the extracellular matrix (ECM) occurs in many epithelial cancers. For example, in ovarian cancer, upregulation of collagen isoform type III has been linked to invasive forms of the disease, and this change may be a potential biomarker. To examine this possibility, we implemented wavelength-dependent second harmonic generation circular dichroism (SHG-CD) imaging microscopy to quantitatively determine changes in chirality in ECM models comprised of different Col I/Col III composition. In these models, Col III was varied between 0 and 40%, and we found increasing Col III results in reduced net chirality, consistent with structural biology studies of Col I and III in tissues where the isoforms comingle in the same fibrils. We further examined the wavelength dependence of the SHG-CD to both optimize the response and gain insight into the underlying mechanism. We found using shorter SHG excitation wavelengths resulted in increased SHG-CD sensitivity, where this is consistent with the electric-dipole-coupled oscillator model suggested previously for the nonlinear chirality response from thin films. Moreover, the sensitivity is further consistent with the wavelength dependency of SHG intensity fit to a two-state model of the two-photon absorption in collagen. We also provide experimental calibration protocols to implement the SHG-CD modality on a laser scanning microscope. We last suggest that the technique has broad applicability in probing a wide range of diseased states with changes in collagen molecular structure.

Figure 1

Representative SHG images and corresponding 3D profiles of Di-8-ANNEPS membrane-stained giant unilamellar vesicles. Parts a and b show examples of acceptable left- and right-circular polarization. Part c is an example of unacceptable circular polarization where black arrows indicate regions of higher intensity lobes due to elliptical polarization. The field of view is 40 × 40 μm².

Figure 2

Representative SHG images of mouse-tail tendon and corresponding CD response: (a) LHCP, (b) RHCP, and (c) CD. Positive and negative CD differences are given in blue and red, respectively. The field of view is 85 × 85 μm². Part d shows the mean normalized SHG-CD data of cleared mouse-tail tendon measured at 780, 830, and 890 nm excitation wavelengths. The asterisk denotes significance p < 0.05. Standard error bars are shown.

Figure 3

Representative SHG images of increasing Col III in Col I gels for LHCP (top) and RHCP (middle) as well as the corresponding CD images (bottom). Positive and negative CD differences are given in blue and red, respectively. The field of view is 85 × 85 μm².

Figure 4

Normalized SHG intensity vs increasing Col III percentage for 780, 830, and 890 nm excitations. Standard error bars are shown.

Figure 5

Mean normalized SHG-CD vs increasing Col III percentage for all three sample sets at 780, 830, and 890 nm excitations. Standard error bars are shown.