Abstract
Polarization dependence second harmonic generation (P-SHG) microscopy is gaining increase popularity for in situ quantification of fibrillar protein architectures. In this report, we combine P-SHG microscopy, new linear least square (LLS) fitting and modeling to determine and convert the complex second-order non-linear optical anisotropy parameter ρ of several collagen rich tissues into a simple geometric organization of collagen fibrils. Modeling integrates a priori knowledge of polyhelical organization of collagen molecule polymers forming fibrils and bundles of fibrils as well as Poisson photonic shot noise of the detection system. The results, which accurately predict the known sub-microscopic hierarchical organization of collagen fibrils in several tissues, suggest that they can be subdivided into three classes according to their microscopic and macroscopic hierarchical organization of collagen fibrils. They also show, for the first time to our knowledge, intrahepatic spatial discrimination between genuine fibrotic and non-fibrotic vessels. CCl4-treated livers are characterized by an increase in the percentage of fibrotic vessels and their remodeling involves peri-portal compaction and alignment of collagen fibrils that should contribute to portal hypertension. This integrated P-SHG image analysis method is a powerful tool that should open new avenue for the determination of pathophysiological and chemo-mechanical cues impacting collagen fibrils organization.
Highlights
Collagens play a central role in the formation of fibrils networks involved in the architecture of tissues and organs
We focus our study on collagen fibrils expansion and remodeling around portal tracts and central veins, which are the site of fibrosis initiation in F1 Metavir stage of liver fibrosis[12]
The third class has microscopic and macroscopic dis-aligned fibrils. These results show that pixel-resolved 3D sub-micrometric organization of collagen fibrils can be well estimated by correlating experimental ρexp and theoretical ρpoiss values taking into account a priori fibrils hierarchical organization without or with fibrillar disorder, considering ubiquitous Poisson noise
Summary
Collagens play a central role in the formation of fibrils networks involved in the architecture of tissues and organs. In extracellular matrixes (ECM), the physical compressive and tensile strains generated by cell traction are key mechanisms involved in the long-range ordering and remodeling of collagen fibrils[1,2] These fibrils, consisting of long and filamentous polymers of collagen molecules, are arrays of axial and lateral supramolecular assembly of quarter-staggered collagen molecules resulting in 67 nm periodic striation observed at ultrastructural level with transmission electron microscopy. SHG has been successfully used for the assessment of liver fibrosis against histological gold standard Fibrosis-Metavir/Ishak staging of patients with moderate to severe fibrosis/cirrhoris[6,7] None of these studies have evaluated ECM fibrillar collagen remodeling despite several reports showing that fibroblast-induced mechanical forces mediate structural rearrangements of ECM collagen fibrils[1,8]. We showed that liver fibrosis characterized by fibrillar remodeling results in reduced fibrillar disorder
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