Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy

Abstract

This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules.

Highly concentrated collagen solutions leading to transparent scaffolds of controlled three-dimensional organizations for corneal epithelial cell colonization

“…7,31 The liquid crystalline samples were also imaged by Second Harmonic Generation microscopy (SHG), which specifically reveals aligned assemblies of collagen molecules or fibrils. 32 We used a custom-built laser-scanning multiphoton microscope as previously described 36 and 2PEF signals were simultaneously detected in two channels equipped with appropriate spectral filters. Power at the sample was typically 3 to 10 mW with 10 μs/pixel acquisition time.…”

“…37 Indeed, the features of frequency responses in oscillatory measurements correspond to those of time responses in creep measurements through Laplace transform as established by the theory of viscoelasticity. We previously checked it 36 for collagen solutions prepared in conditions 4 with viscoelastic moduli scaling as ~  and strain under load (scaling as ~ t…”

Electrospinning of pure, native, cross-linker free self-supported collagen membrane

“…The structural specificity of SHG images has been further improved by acquiring polarization-resolved images (pSHG), which measures the main orientation of collagen in the imaging plane, in every pixel of the SHG image. 30 Given that each color codes for a given direction, the presence of dots with many different colors throughout the whole image shows that the highly packed structures of native collagen are randomly distributed with respect to one another (Fig. 3A2).…”

“…Transdetection was used for hydrated sample, with no binning. pSHG images were processed as already described 30 to provide two images: (i) the average of all images acquired with the set of linear polarizations, that is similar to an SHG image acquired with circular polarization; (ii) a map of the main orientation of collagen in the image plane. The latter pSHG image is displayed using the HSV Look-up-table, where H is the orientation displayed in the insert, and V is the brightness, which is set to 1 if the pSHG processing is satisfactory (R2>0.5).…”

Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases

“…This structure has been validated by P-SHG and numerical calculations. 39 The characteristic pattern of a plywood mesophase is present up to a gelatin ratio of 20%. From 25% to 50% no clear textures are seen.…”