Determination of collagen fibril size via absolute measurements of second-harmonic generation signals
Nat Commun. 2014-09-16; 5(1):
DOI: 10.1038/ncomms5920
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Bancelin S(1), Aimé C(2), Gusachenko I(1), Kowalczuk L(3), Latour G(1), Coradin T(2), Schanne-Klein MC(1).
Author information:
(1)Laboratoire d’Optique et Biosciences, Ecole Polytechnique, CNRS, INSERM U696, Route de Saclay, 91128 Palaiseau, France.
(2)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.
(3)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.
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.