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.

Probing ECM remodeling in idiopathic pulmonary fibrosis via second harmonic generation microscopy analysis of macro/supramolecular collagen structure

“…Imaging was performed with a fundamental laser wavelength of 890 nm for SHG forward-backward (F/B) and P-SHG analysis and 780 nm for SHG-CD; the shorter wavelength for the latter provides greater sensitivity. 5 Average powers at the focus were ∼30 to 50 mW using a 40× 0.8 NA water immersion lens (LUMPlanFL; Olympus, Tokyo, Japan) and a 0.9 NA condenser. The resulting lateral and axial resolutions were ∼0.7 and 2.5 μm, respectively.…”

“…14 The linear and circular polarization states were validated as previously described by imaging cylindrically symmetric giant vesicles. 5,14 The polarization control was also run by a custom LabVIEW program interfaced to the FluoView scanning system.…”

“…SHG-CD analysis was used to interrogate the overall chirality of the human lung tissues; the method has been described previously. 5 In brief, images of IPF and normal lung tissues were taken using left-and right-handed (LH and RH) circularly polarized (CP) laser excitation and were obtained 30-μm deep into the optically cleared tissues to avoid boundary effects. To account for variations in intensity in the different samples, we report the normalized SHG-CD response defined as E Q -T A R G E T ; t e m p : i n t r a l i n k -; e 0 0 2 ; 1 1 6 ; 6 0 7…”

“…2]. 5 Representative SHG-CD images are shown in Fig. 6(a) for both normal and IPF, where red and blue correspond to the sign of the response and corresponds to the polarity of the fiber and the magnitude arises from the alignment of the triple helices within the focal volume.…”

“…For example, SHG polarization-resolved methods [e.g., linear SHG polarization analysis (P-SHG) and circular dichroism (SHG-CD)] can extract information on the helical structure of collagen (e.g., α-pitch angle and triple helical chirality). 5 For example, we previously showed that collagen molecular aspects were different in human ovarian cancer from normal ovarian tissues. 6 This is also relevant for IPF as there have been earlier reports using immunostaining that showed the Col I and Col III isoform balance changes during disease progression and is also different from that of normal tissue.…”