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Second harmonic generation in skin and cornea



Marie-Claire Schanne-Klein / DR CNRS
M. Schmeltz / PhD student
C. Raoux / PhD student
Close collaboration with Gaël Latour / Paris-Saclay University

Internship project: SHG imaging of pathological corneas / Proposition de stage : Imagerie SHG de cornées pathologiques


Second Harmonic Generation (SHG) microscopy is an efficient technique to probe the 3D architecture of collagen, which is the main structural protein in mammals and determines the architecture of many tissues (skin, cornea, artery, bone…).  Our main recent results are the following:

- We deciphered the physical origin of SHG signal from collagen at the molecular and fibrillar scales and we measured quantitatively this response.
- We implemented polarization-resolved SHG microscopy and showed that this technique probes the fibrils orientation and the orientational disorder at sub-µm scale within tissues.
- We assessed the progression of fibrotic pathologies in lung and renal tissues and characterized the 3D organization of collagen-based tissue substitutes.
- We characterized multimodal nonlinear optical signals in Human corneas and demonstrated in vivo structural imaging of rat corneas.
- We combined SHG imaging and mechanical assays to decipher the relationships between macroscopic tissue response and microscopic structure in skin and cornea.
- We correlated multiphoton microscopy to Infra-Red nanoscopy to characterize the denaturation of collagen to gelatin (see also application to parchments degradation).

Highlights / review articles

[French] Mesurer optiquement le diamètre des fibrilles de collagène, Actualités scientifiques de l’INP CNRS
[English] SHG imaging and quantization of fibrosis, Schanne-Klein M.-C., In "Second Harmonic Generation Imaging", F. S. Pavone and P. J. Campagnola Editors, p.349-371 (Taylor and Francis, 2013)

Main ongoing collaborations

- G. Latour, IMNC (CNRS, Université Paris-Sud - Orsay)
- G. Mosser, C. Aimé et T. Coradin, LCMCP (CNRS, UPMC, Collège de France - Paris)
- J.-M. Allain, LMS (CNRS, Ecole Polytechnique - Palaiseau)
- F. Ruggiero, C. Bonod-Bidaud, IGFL (CNRS, ENS-Lyon)
- L. Robinet, CRC (CNRS, Museum National d’Histoire Naturelle - Paris)
- A. Dazzi, A. Deniset-Besseau, LCP (CNRS, Université Paris-Sud - Orsay)
- V. Borderie, K. Grieve, D. Ghoubay, Hôpital des 15-20, Paris
- F. Légaré, INRS, Montréal, Canada

Recent publications

How aging impacts skin biomechanics: a multiscale study in mice, B. Lynch, C. Bonod-Bidaud, G. Ducourthial, J.-S. Affagard, S. Bancelin, S. Psilodimitrakopoulos, F. Ruggiero, J.-M. Allain, and M.-C. Schanne-Klein, Scientific Reports 7, 13750 (2017).     

Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea, D. Ghoubay-Benallaoua, C. de Sousa, R. Martos, G. Latour, M.-C. Schanne-Klein, E. Dupin, V. Borderie, Plos One 12 (11), e0188398 (2017).

Stromal striae: a new insight into corneal physiology and mechanics, K. Grieve, D. Ghoubay, C. Georgeon, G. Latour, A. Nahas, K. Plamann, C. Crotti, R. Bocheux, M. Borderie, T.-M. Nguyen, F. Andreiuolo, M.-C. Schanne-Klein, and V. Borderie, Scientific Reports 7, 13584 (2017).       

Stabilization of collagen fibrils by gelatin addition: a study of collagen/gelatin dense phases, F. Portier, C. Teulon, A. Nowacka-Perrin, F. Guenneau, M.-C. Schanne-Klein, and G. Mosser, Langmuir 33 (45), 12916–12925 (2017).

A novel microstructural interpretation for the biomechanics of mouse skin derived from multiscale characterization, B. Lynch, S. Bancelin, C. Bonod-Bidaud, J.-B. Guesquin, F. Ruggiero, M.-C. Schanne-Klein, and J.-M. Allain, Acta Biomateriala 50, 302-311 (2017).

Cell Viability and Shock Wave Amplitudes in the Endothelium of Porcine Cornea exposed to Ultrashort Laser Pulses, S. A. Hussain, C. Milián, C. Crotti, L. Kowalczuk, F. Alahyane, Z. Essaïdi, A. Couairon, M.-C. Schanne-Klein, and K. Plamann, Graefes Arch. Clin. Exp. Ophthalmol. 255, 945-953 (2017). doi:10.1007/s00417-017-3583-3

Affine kinematics in planar fibrous connective tissues: an experimental investigation, C. Jayyosi, J.-S. Affagard, G. Ducourthial, C. Bonod-Bidaud, B. Lynch, S. Bancelin, F. Ruggiero, M.-C. Schanne-Klein, J.-M. Allain, K. Bruyère-Garnier, and M. Coret, Biomechanics and Modeling in Mechanobiology 16 (4), 1459-1473 (2017).

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy, R. Genthial, E. Beaurepaire, M.-C. Schanne-Klein, F. Peyrin, D. Farlay, C. Olivier, Y. Bala, G. Boivin, J.-C. Vial, J. Derouard, D. Débarre, A. Gourrier, Scientific Reports, 7 :3419 (2017). doi:10.1038/s41598-017-03548-5

Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments, G. Latour, L. Robinet, A. Dazzi, F. Portier, A. Deniset-Besseau, and M.-C. Schanne-Klein, Sci. Rep. 6, 26344 (2016).

Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation, N. Vuillemin, P. Mahou, D. Débarre, T. Gacoin, P.-L. Tharaux, M.-C. Schanne-Klein, W. Supatto, and E. Beaurepaire, Sci. Rep. 6, 29863 (2016). Erratum: 6, 31258 (2016)

Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy, C. Teulon, A. Tidu, F. Portier, G. Mosser, and M.-C. Schanne-Klein, Opt. Express 24, 16084 (2016).

Simultaneous microstructural and mechanical characterization of human corneas at increasing pressure, A. Benoit, G. Latour, M.-C. Schanne-Klein, and J.-M. Allain, J. Mech. Behav. Biomed. Mater. 60, 93-105 (2016)

Ex vivo multiscale quantitation of skin biomechanics in wild-type and genetically-modified mice using multiphoton microscopy, S. Bancelin, B. Lynch, C. Bonod-Bidaud, G. Ducourthial, S. Psilodimitrakopoulos, P. Dokladal, J.-M. Allain, M.-C. Schanne-Klein, and F. Ruggiero, Scientific Reports 5, 17635 (2015)

Optimization of Picrosirius Red Staining Protocol to Determine Collagen Fiber Orientations in Vaginal and Uterine Cervical Tissues by Mueller Polarized Microscopy, A. Nazac, S. Bancelin, B. Teig, B. H. Ibrahim, H. Fernandez, M.-C. Schanne-Klein, A. De Martino, Microsc. Res. Tech. 78, 723-730 (2015)

Theoretical, numerical and experimental study of geometrical parameters that affect anisotropy measurements in polarization-resolved SHG microscopy, C. Teulon, I. Gusachenko, G. Latour, and M.-C. Schanne-Klein, Opt. Express 23, 9313-9328 (2015).

Determination of collagen fibril size via absolute measurements of second-harmonic generation signals, S. Bancelin, C. Aimé, I. Gusachenko, L. Kowalczuk, G. Latour, T. Coradin, M.-C. Schanne-Klein, Nat. Commun. 5, art. 4920 (2014)

Fibrillogenesis from nanosurfaces: multiphoton imaging and stereological analysis of collagen 3D self-assembly dynamics, S. Bancelin, E. Decencière, V. Machairas, C. Albert, T. Coradin, M.-C. Schanne-Klein, C. Aimé, Soft Matter 10, 6651 (2014)

Determination of collagen fiber orientation in histological slides using Mueller microscopy and validation by second harmonic generation imaging, S. Bancelin, A. Nazac, B. H. Ibrahim, P. Dokladal, E. Decencière, B. Teig, H. Haddad, H. Fernandez, M.-C. Schanne-Klein, A. de Martino, Opt. Express 22, 22561 (2014)

Achievement of cornea-like organizations in dense collagen I solutions: clues to the physico-chemistry of cornea morphogenesis, P. De Sa Peixoto, A. Deniset-Besseau, M. Schmutz, A. Anglo, C. Illoul, M.-C. Schanne-Klein, G. Mosser, Soft Matter 9, 11241 (2013)

Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication, R. Lestini, S. P. Laptenok, J. Kühn, M. A. Hink, M.-C. Schanne-Klein, U. Liebl, H. Myllykallio, Nucleic Acid Res. 22, 10358 (2013)

Numerical simulation of polarization-resolved second harmonic microscopy in birefringent media I. Gusachenko and M.-C. Schanne-Klein, Phys. Rev. A 88, 053811 (2013)

A Bottom-Up Approach to Build the Hyperpolarizability of Peptides and Proteins from their Amino-Acids, J. Duboisset, A. Deniset-Besseau, E. Benichou, I. Russier-Antoine, N. Lascoux, C. Jonin, F. Hache, M.-C. Schanne-Klein, P.-F. Brevet, J. Phys. Chem. B 117, 9877 (2013)

Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas, L. Kowalczuk, G. Latour, J.-L. Bourges, M. Savoldelli, J.-C. Jeanny, K. Plamann, M.-C. Schanne-Klein, F. Behar-Cohen, Trans. Vis. Sci. Tech. 2, 3 (2013)

Probing ordered lipid assemblies with polarized Third-Harmonic-Generation microscopy, M. Zimmerley, P. Mahou, D. Debarre, M.-C. Schanne-Klein, E. Beaurepaire, Phys. Rev. X 3, 011002 (2013)

Hyperglycemia-Induced Abnormalities in Rat and Human Corneas: The Potential of Second Harmonic Generation Microscopy, G. Latour, L. Kowalczuk, M. Savoldelli, J.-L. Bourges, K. Plamann, F. Behar-Cohen, M.-C. Schanne-Klein, PLoS ONE 7, e48388 (2012) pdf

In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy, G. Latour, J.-P. Echard, M. Didier, M.-C. Schanne-Klein, Opt. Express 20, 24623 (2012) pdf

3D resolved mapping of optical aberrations in thick tissues, J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, Biomed. Opt. Express 3, 1898 (2012) pdf

Imaging and 3D Morphological Analysis of Collagen Fibrils, H. Altendorf, E. Decencière, D. Jeulin, P. D. S. Peixoto, A. Deniset-Besseau, E. Angelini, G. Mosser, M.-C. Schanne-Klein, J. Microscopy 247, 161 (2012).

In situ three-dimensional monitoring of collagen fibrillogenesis using SHG microscopy, S. Bancelin, C. Aimé, T. Coradin, M.-C. Schanne-Klein, Biomed. Opt. Express 3, 1446 (2012). pdf

Polarization-resolved second-harmonic microscopy in tendon upon mechanical stretching, I. Gusachenko, Y. G. Houssen, V. Tran, J.-M. Allain, M.-C. Schanne-Klein, Biophys. J. 102, 2220 (2012). preprint

In vivo structural imaging of the cornea by polarization-resolved Second Harmonic microscopy, G. Latour, I. Gusachenko, L. Kowalczuk, I. Lamarre, M.-C. Schanne-Klein, Biomed. Opt. Express 3, 1 (2012). pdf

Quantitative assessment of collagen I liquid crystal organizations: role of ionic force and acidic solvent, and evidence of new phases, P. De Sa Peixoto, A. Deniset-Besseau, M.-C. Schanne-Klein, G. Mosser, Soft Matter 7, 11203 (2011).

Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic generation microscopy, Y. Goulam Houssen, I. Gusachenko, M.-C. Schanne-Klein, J.-M. Allain, J. Biomech. 44, 2047 (2011) preprint [PDF - 696 Ko]

Multiphoton microscopy of engineered dermal substitutes: assessment of 3D collagen matrix remodeling induced by fibroblasts contraction, A.-M. Pena, D. Fagot, C. Olive, J.-F. Michelet, J.-B. Galey, F. Leroy, E. Beaurepaire, J.-L. Martin, A. Colonna, M.-C. Schanne-Klein, J. Biomed. Opt. 15, 056018 (2010) pdf

Polarization-resolved Second Harmonic microscopy in thick anisotropic tissues, I. Gusachenko, G. Latour, M.-C. Schanne-Klein, Opt. Express 18, 19339 (2010) pdf [PDF - 1 Mo]

Multimodal nonlinear imaging of the human cornea, F. Aptel, N. Olivier, A. Deniset-Besseau, J.-M. Legeais, K. Plamann, M.-C. Schanne-Klein, E. Beaurepaire, Invest. Ophthalmol. Vis. Sci 51, 2459 (2010) postprint

Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea, N. Olivier, F. Aptel, K. Plamann, M.-C. Schanne-Klein, E. Beaurepaire, Opt. Express 18, 5028 (2010)

Nonlinear optical imaging of lyotropic cholesteric liquid crystals A. Deniset-Besseau et al, Opt. Express 18, 1113 (2010)

Measurement of the Second-Order Hyperpolarizability of the Collagen Triple helix and Determination of its Physical origin A. Deniset-Besseau et al, J. Phys. Chem. B 113, 13437 (2009) pdf [PDF - 695 Ko]

Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling M. Strupler et al, J. Biomed. Optics 13, 054041 (2008) PDF

Second harmonic imaging and scoring of collagen in fibrotic tissues M. Strupler et al, Opt. Express 15, 4054 (2007) PDF Movie

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy A.-M. Pena et al, Microsc. Res. Tech. 70, 162 (2007) PDF

Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure T. Boulesteix et al, Cytometry 69A, 20 (2006) PDF

Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy A.-M. Pena et al, Opt. Express 13, 6268 (2005); 13, 6667 (2005) PDF

Chiroptical effects in the second harmonic signal of collagens I and IV A.-M. Pena et al, J. Am. Chem. Soc. 127, 10314 (2005) PDF

Second harmonic microscopy of abstract living cardiac myocytes: measurements of sarcomere length with 20 nm accuracy T. Boulesteix et al, Opt. Lett. 29, 2031 (2004) PDF



PhD students
- Thierry Boulesteix : Microscopie non linéaire: chiralité et génération de second harmonique (07/2004) - Professeur en classes préparatoires
- Ana-Maria Pena : Génération de seconde harmonique par le collagène et application à l'étude de fibroses par microscopie multiphoton (09/2006) - Research Engineer at L'Oreal-Advanced research
- M. Strupler : Imagerie du collagène par microscopie multiphotonique. Application aux fibroses rénales (11/2008) - Research Associate at Polytechnique Montreal
- Ivan Gusachenko : Polarization-resolved Second Harmonic Microscopy in collagenous tissues (10/2012) - Post-doc position at St Andrews, UK
- Stéphane Bancelin : Imagerie quantitative du collagène par imagerie de seconde harmonique (12/2013) - Post-doc position at Bordeaux, France
- Claire Teulon: Imagerie quantitative du collagène par microscopie par génération de second harmonique résolue en polarisation (10/2016) - Post-doc position at EPFL, Suisse

- Ariane Deniset-Besseau, 2008-2009 - Associate professor at Paris-Sud University
- Yannick Goulam Houssen, 2009 - Research Engineer at Paris 5 University
- Gaël Latour, 2010-2012 - Associate professor at Paris-Sud University
- Sotiris Psilodimitrakopoulos, 2013-2014 - Foundation for Research and Technology, Irákleion, Greece
- Guillaume Ducourthial, 2015-2016 - Post-doc position at Boston, USA