Determination of collagen fibril size via absolute measurements of second-harmonic generation signals

Abstract

The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonicelectron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea.

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation

“…Based on these quantitative assessments, we conclude that the SHG can be ascribed to a filamentous ultrastructure assembled from R234 domains, but not to unassembled monomeric R234 itself. Complete assignment and determination of the filament diameter could be performed by absolute measurement of SHG signals44.…”

Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry

“…The presence of strong sample birefringence can affect the relative magnitude of the unique tensor elements, which can complicate quantitative polarization-dependent SHG analyses (62). As the collagen thickness approaches and exceeds the forward coherence length, interference effects have the potential to impact the overall intensity of the observed SHG (57,63). Therefore, all analysis was based exclusively on the polarization-dependence derived from the relative intensities.…”

L’imagerie multiphoton des peaux naturelles et synthétiques. Un nouvel outil pour l’évaluation des produits cosmétiques

“…Ces liaisons peptidiques constituent le squelette de toutes les protéines, mais seule une protéine de structure fibrillaire comme le collagène permet de conserver la non centro-symétrie à l'échelle micrométrique. La forte densité des fibres de collagène permet de plus d'obtenir un signal SHG relativement important et donc de détecter des fibrilles de collagène d'un diamètre aussi petit que 30 nm [4] ! L'imagerie SHG est ainsi de plus en plus utilisée pour imager l'organisation 3D des fibres de collagène dans le derme de peaux naturelles ou reconstruites (figure 2).…”