We use SHG microscopy to study collagen alterations in idiopathic pulmonary fibrosis (IPF) in human tissues and in vitro models. Texture analysis successfully classified the fiber morphology in normal and IPF tissues at near 100% accuracy. SHG polarization measurement indicated reduced chirality in the collagen triple helix in IPF in both tissues and spheroid models. We have created a “collagen atlas” in normal and IPF lung using the combination of SHG and THG to image large areas in annotated histology slides. The combined collagen and cellular organization data formed an accurate classification scheme and identified the most sensitive features.
We use SHG microscopy to study collagen alterations in idiopathic pulmonary fibrosis (IPF) in human tissues and in vitro models. Texture analysis successfully classified the fiber morphology in normal and IPF tissues at near 100% accuracy. SHG polarization measurement indicated reduced chirality in the collagen triple helix in IPF in both tissues and spheroid models. Using SHG directional measurements (F/B), we found the collagen fibril and macromolecular structures are altered in IPF, consistent with decreased organization. We are developing a rigorous phasematching approach to determine the collagen fibril size, packing, and polarity based on these measurements.
Significance: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored.
Aim: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition).
Approach: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids.
Results: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group.
Conclusions: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.
We use SHG microscopy to study the collagen alterations in idiopathic pulmonary fibrosis (IPF) in ex vivo human tissues and in vitro spheroid models. We found the collagen fibril and macromolecular structures are altered in IPF, consistent with decreased organization. Further, machine learning based texture analysis successfully classified the fiber morphology at near 100% accuracy. To gain insight into the underlying mechanisms of these alterations, we grew IPF based spheroids under different crosslinking conditions (increased or inhibited). SHG polarization measurements (linear and SHG-CD) indicated that increased crosslinking altered the collagen helical structure similar to those of the human tissues.
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