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

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

Keywords: collagen; fibrosis; polarization; scattering; second harmonic generation.

Fig. 1

Combined collagen and $\alpha$-SMA staining in normal [(a) and (c)] and IPF [(b) and (d)] tissues. The top row gives SHG images only and the bottom row is an overlap of SHG (grayscale) and TPEF (red) for $\alpha$-SMA, identifying fibrotic regions. $\mathrm{S}\text{cale bar}=30\mu \mathrm{m}$.

Fig. 2

Collagen concentration data of normal (blue) and IPF (red) lung tissues. Standard error bars are shown. Number of samples were six for both IPF and normal with three separate slices in each case. Note: * indicates $p<0.05$.

Fig. 3

Spectral dependence of ${\mu }_s^{\prime }$ over UV/Vis and NIR wavelengths for normal and IPF tissues where the fit is to the Whittle–Matérn correlation function. The IPF tissues are more highly scattering but have a stronger spectral slope (lower $m$), indicting a broader range of scatter sizes. There were $\sim 20$ independent measurements at each wavelength using the different tissues.

Fig. 4

Local analysis of the SHG emission directionality. (a) Measured F/B as a function of depth into normal (blue) and IPF (red) tissues; solid and open symbols correspond to measured and simulated responses, respectively. The resulting $F_{\mathrm{SHG}}/B_{\mathrm{SHG}}$ in $15\times 15\text{\hspace{0.17em}\hspace{0.17em}pixel}$ patches for (b) normal and (c) IPF tissues. Number of stacks were 34 and 75 for IPF and normal, respectively. $\text{Scalebar}=30\mu \mathrm{m}$.

Fig. 5

Linear polarization analysis of normal (blue) and IPF (red) tissues. (a) The reconstructed pixel-based response; (b) the extracted pitch angles. The data were similar to each other, inconsistent with an increase in Col III abundance in IPF.

Fig. 6

(a), (b) Normalized SHG-CD data of cleared normal (blue) and IPF (red) lung tissues measured at 780-nm excitation wavelength. (a) The red and blue correspond to positive and negative SHG-CD values, respectively, which are determined by the fiber polarity. (b) Standard error bars are shown. Number of unique images were 134 and 121 for IPF and normal, respectively. $\text{Field size}=85\times 85\mu \mathrm{m}$. Note: **** represents $p<0.00001$.