Abstract
Second-harmonic generation (SHG) microscopy is currently the preferred technique for visualizing collagen in intact tissues, but the usual implementations struggle to reveal collagen fibrils oriented out of the imaging plane. Recently, an advanced SHG modality, circular dichroism SHG (CD-SHG), has been proposed to specifically highlight out-of-plane fibrils. In this study, we present a theoretical analysis of CD-SHG signals that goes beyond the electric dipolar approximation to account for collagen chirality. We demonstrate that magnetic dipolar contributions are necessary to analyze CD-SHG images of human cornea sections and other collagen-rich samples. We show that the sign of CD-SHG signals does not reveal whether collagen fibrils point upwards or downwards as tentatively proposed previously. CD-SHG instead probes the polarity distribution of out-of-plane fibril assemblies at submicrometer scale, namely homogeneous polarity versus a mix of antiparallel fibrils. This makes CD-SHG a powerful tool for characterizing collagen organization in tissues, specifically the degree of disorder, which is affected during pathological remodeling. CD-SHG may thus serve to discriminate healthy and diseased collagen-rich tissues.
Highlights
Collagen is the most abundant protein in mammals and a major component of connective tissues such as arteries, skin, bones and cornea
This study aims at deriving a theoretical analysis of circular dichroism (CD)-Second-harmonic generation (SHG) signals that includes magnetic dipolar contributions and at elucidating what collagen structural information is provided by circular dichroism SHG (CD-SHG) imaging
The down image is digitally flipped with respect to the same axis to facilitate the comparison between the two images: the out-of-plane angle is opposite ψdown = −ψup, while the in-plane angle is unchanged φdown = φup. These up/down experiments were conducted on various thin collagen-rich samples: six transverse sections of human corneas (18 CD-SHG images in total), in vitro isolated collagen fibrils, and collagen fibrillar membranes on a first setup and a section of murine fascia on a second setup (Supplement 1.2)
Summary
Collagen is the most abundant protein in mammals and a major component of connective tissues such as arteries, skin, bones and cornea. Fibrillar collagens are characterized by a hierarchical structure: triple helix molecules self-assemble into fibrils with 12 to > 500 nm diameter that further form various tridimensional (3D) structures at larger scales [1]. This 3D multiscale structure is a key distinctive feature of every tissue that governs its functional behavior, notably its mechanical properties [2]. In situ 3D imaging of collagen is a major biomedical concern to
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