title: 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-harmonic-electron 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.
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Route de Saclay, 91128 Palaiseau, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bancelin
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire de Chimie de la Matière Condensée de Paris, Collège de France, 11 place Marcelin Berthelot, 75231 Paris, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Aimé
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Route de Saclay, 91128 Palaiseau, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Gusachenko
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS 872, Team17: Physiopathology of Ocular Diseases, Therapeutic Innovations, Centre de Recherche des Cordeliers, 15 rue de l'école de médecine, 75006 Paris, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Kowalczuk
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Route de Saclay, 91128 Palaiseau, France.
authors.forename: Gaël
authors.initials: G
authors.lastname: Latour
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, CNRS, Laboratoire de Chimie de la Matière Condensée de Paris, Collège de France, 11 place Marcelin Berthelot, 75231 Paris, France.
authors.forename: Thibaud
authors.initials: T
authors.lastname: Coradin
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Route de Saclay, 91128 Palaiseau, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Schanne-Klein
mesh_terms: D000818:Animals; D024042:Collagen Type I / Q000302:isolation & purification / Q000648:ultrastructure*; D003886:Descemet Membrane / Q000473:pathology / Q000648:ultrastructure*; D003921:Diabetes Mellitus, Experimental / Q000473:pathology; D005109:Extracellular Matrix / Q000737:chemistry / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted / Q000706:statistics & numerical data*; D008854:Microscopy, Electron / Q000295:instrumentation* / Q000379:methods; D051381:Rats; D013623:Tail / Q000737:chemistry
chemical_list: D024042:Collagen Type I