Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation

International audiencePolarization resolved second harmonic generation (PSHG) is developed to study, at the microscopic scale, the impact of aging on the structure of type I collagen fibrils in two-dimensional coatings. A ribose-glycated collagen is also used to mimic tissue glycation usually described as an indicator of aging. PSHG images are analyzed using a generic approach of the molecular disorder information in collagen fibrils, revealing significant changes upon aging, with a direct correlation between molecular disorder and fibril diameters

In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography, Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model

Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4–8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success

Microscopie polarimétrique du collagène de type I par génération de second harmonique dans des systèmes modèles et tissus

La génération de second harmonique (SHG) est un processus non-linéaire qui se produit dans des structures non-centrosymétriques, comme c'est le cas de certains matériaux cristallins ou biologiques. Il consiste à coupler deux champs à la même fréquence pour générer un photon à la fréquence double. La manipulation de la polarisation des champs incidents donne accès à des informations microscopiques et structurales de l'échantillon. De plus, l'utilisation d'une détection polarisée permet de mettre à jour des effets physiques dans les assemblages moléculaires biologiques.Dans ces travaux de thèse, nous nous intéressons principalement à l'étude des fibres de collagène I en SHG polarisée dans des échantillons modèles et des tissus. Nous étendons cette étude à la compréhension de l'interaction des fibres avec leur environnement cellulaire pour ensuite, aborder la problématique des tissus cancéreux. Enfin, nous proposons différents modèles microscopiques de la structure du collagène, évalués par une méthode basée sur la décomposition en série de Fourier du signal polarisé, pour apporter un diagnostic quantitatif sur des échantillons biologiques.Second harmonic generation (SHG) is a non-linear process which consists in coupling two photons at the same frequency to generate one photon at the twice frequency. It generally occurs in non-centrosymmetric samples such as crystals or molecular assemblies. The manipulation of the optical field polarization gives access to structural and microscopic informations. Moreover, using polarized detection allows to determine physical effects in molecular assemblies.In this Phd thesis, we are particulary interested in studying polarized SHG signals from collagen type I fibers in model samples and tissues. We extend our work to the investigation of the interaction of the fibers with their cellular environment. We also address the problematic of cancerous tissues.Finally, we propose several models for the microscopic structure of collagen, evaluated by a method based on the Fourier decomposition of the polarized SHG signal, to provide a quantitative diagnosis of biological samples

Microscopie polarimétrique du collagène de type I par génération de second harmonique dans des systèmes modèles et tissus

La génération de second harmonique (SHG) est un processus non-linéaire qui se produit dans des structures non-centrosymétriques, comme c'est le cas de certains matériaux cristallins ou biologiques. Il consiste à coupler deux champs à la même fréquence pour générer un photon à la fréquence double. La manipulation de la polarisation des champs incidents donne accès à des informations microscopiques et structurales de l'échantillon. De plus, l'utilisation d'une détection polarisée permet de mettre à jour des effets physiques dans les assemblages moléculaires biologiques.Dans ces travaux de thèse, nous nous intéressons principalement à l'étude des fibres de collagène I en SHG polarisée dans des échantillons modèles et des tissus. Nous étendons cette étude à la compréhension de l'interaction des fibres avec leur environnement cellulaire pour ensuite, aborder la problématique des tissus cancéreux. Enfin, nous proposons différents modèles microscopiques de la structure du collagène, évalués par une méthode basée sur la décomposition en série de Fourier du signal polarisé, pour apporter un diagnostic quantitatif sur des échantillons biologiques.Second harmonic generation (SHG) is a non-linear process which consists in coupling two photons at the same frequency to generate one photon at the twice frequency. It generally occurs in non-centrosymmetric samples such as crystals or molecular assemblies. The manipulation of the optical field polarization gives access to structural and microscopic informations. Moreover, using polarized detection allows to determine physical effects in molecular assemblies.In this Phd thesis, we are particulary interested in studying polarized SHG signals from collagen type I fibers in model samples and tissues. We extend our work to the investigation of the interaction of the fibers with their cellular environment. We also address the problematic of cancerous tissues.Finally, we propose several models for the microscopic structure of collagen, evaluated by a method based on the Fourier decomposition of the polarized SHG signal, to provide a quantitative diagnosis of biological samples.AIX-MARSEILLE3-Bib. élec. (130559903) / SudocSudocFranceF

Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation

International audienceIn this work we investigate a generic method able to extract information on molecular organization in biological samples from polarized second harmonic generation (SHG) microscopy, without the need to infer an a priori model for the molecular orientational distribution. The mean orientation of this distribution, as well as its first and third orders of symmetry, are estimated by monitoring SHG intensity signals under a varying incident polarization. We introduce, in particular, a reduction of the problem to a two-dimensional approach appropriate to the microscopy geometry. This method allows us to retrieve determining information which is not available in the traditional model-oriented methods, as illustrated in molecular-order imaging in collagen fibrils. The precision of the parameters estimation is evaluated by a Monte Carlo analysis, based on the Poisson noise statistics of the measured signal

Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging

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Quantifying molecular order in biological samples using polarimetric muti-modal nonlinear microscopy

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Complete histological evaluation of the cervix.

<p>Mucous plug (m), epithelial cells (e), mucosal chorion (c), submucosa (s).</p