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Third harmonic (THG) microscopy

Nonlinear microscopies have the unique ability to provide micrometer-scale 3D images from within complex, scattering samples like biological tissues.

In particular, third-harmonic generation (THG) microscopy detects interfaces and optical heterogeneities and provides 3D structural images of unstained biological samples. This information can be combined with other nonlinear signals such as 2PEF and SHG.

Since THG is a coherent process, signal generation must be properly analyzed in order to interpret the images. We study the contrast mechanisms in THG microscopy (phase matching, nonlinear susceptibilities), and we develop novel applications such as :
• imaging morphogenesis in small animal models (zebrafish, drosophila);
• imaging lipids in cells and tissues;
• polarization-resolved THG analysis of organized media: human cornea, skin lipids, etc


Multimodal nonlinear imaging of intact excised human corneas. Adapted from IOVS 2010 and Opt Express 2010.


THG imaging of Drosophila embryo morphogenesis. Adapted from Opt Lett 29, 2881, 2004. Right: combined THG/2PEF imaging of a GFP-labeled Drosophila embryo. THG (orange) reveals lipid droplets. Adapted from Nature Methods 2006.

 

Related publications

High-speed polarization-resolved third-harmonic microscopy
J. Morizet, G. Ducourthial, W. Supatto, A. Boutillon, R. Legouis, M.-C. Schanne-Klein, C. Stringari, E. Beaurepaire
Optica 6 (3), 385-388 (2019)
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, D. Débarre, A. Gourrier
Scientific Reports 7, 3419 (2017).
Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0–1.2 µm wavelength range
D. Débarre, N. Olivier, W. Supatto, E. Beaurepaire
PLoS ONE 9(8): e104250 (2014).
Probing ordered lipid assemblies with polarized third-harmonic-generation microscopy
M. Zimmerley, P. Mahou, D. Débarre, M.-C. Schanne-Klein & E. Beaurepaire
Phys. Rev. X 3, 011002 (2013).
Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy
T. Chang, M. Zimmerley, K.P. Quinn, I. Lamarre-Jouenne, D.L. Kaplan, E. Beaurepaire & I. Georgakoudi
Biomaterials 34(34) 8607-16 (2013).
Third-harmonic generation microscopy with Bessel beams: a numerical study
N. Olivier, D. Débarre, P. Mahou, and E. Beaurepaire
Opt. Express 20(22), 24886-24902 (2012).
Multicolor two-photon tissue imaging by wavelength mixing
P. Mahou, M. Zimmerley, K. Loulier, K. Matho, G. Labroille, X. Morin, W. Supatto, J. Livet, D. Débarre & E. Beaurepaire
Nature Methods 9(8) 815-818 (2012).
Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos
P. Mahou, N. Olivier, G. Labroille, L. Duloquin, J-M. Sintes, N. Peyriéras, R. Legouis, D. Débarre, and E. Beaurepaire
Biomed. Opt. Express 2(10), 2837-2849 (2011).
Advances in multiphoton microscopy for imaging embryos
W. Supatto, T.V. Truong, D. Debarre, and E. Beaurepaire
Current Opinion in Genetics & Development 21(5) 538-548(2011).
Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy
N. Olivier, M. Luengo-Oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras & E. Beaurepaire
Science 339(5994), 967-71 (2010).
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. Ophtalmol. Vis. Sci. 51(5), 2459-65 (2010). Cover.
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(5), 5028-40 (2010).
Dynamic aberration correction for multiharmonic microscopy
N. Olivier, D. Débarre & E. Beaurepaire
Opt. Lett. 34(20), 3145-7 (2009).
Third-harmonic generation microscopy with focus-engineered beams: a numerical study
N. Olivier & E. Beaurepaire
Opt. Express 16(19), 14703-14715 (2008). PDF.
Signal epidetection in third-harmonic generation microscopy of turbid media
D. Débarre, N. Olivier & E. Beaurepaire
Opt. Express 15 (14) 8913-24 (2007). PDF.
Quantitative characterization of biological liquids for third-harmonic generation microscopy
D. Débarre & E. Beaurepaire
Biophys. J. 92(2), 603-612 (2007). PDF.
Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy
D. Débarre, W. Supatto, A.-M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M.-C. Schanne-Klein & E. Beaurepaire
Nature Methods 3(1), 47-53 (2006). PDF.
Structure sensitivity in third-harmonic generation microscopy
D. Débarre, W. Supatto & E. Beaurepaire
Opt. Lett. 30(16), 2134-2136 (2005). PDF.
Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos
D. Débarre, W. Supatto, E. Farge, B. Moulia, M.-C. Schanne-Klein & E. Beaurepaire
Opt. Lett. 29(24), 2881-2883 (2004). PDF.
In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses
W. Supatto, D. Débarre, B. Moulia, E. Brouzés, J.-L. Martin, E. Farge & E. Beaurepaire
PNAS 102(2), 1047-1052 (2005). PDF.

Book chapter
THG microscopy of cells and tissues: contrast mechanisms and applications
N. Olivier, D. Débarre & E. Beaurepaire,
in Series in Cellular and Clinical Imaging, SHG Imaging pp. 51-80, Taylor & Francis 2013.
Introduction article (French)
Microscopies multi-harmoniques pour l'imagerie structurale de tissus intacts
D. Débarre, A-M. Pena, W. Supatto, T. Boulesteix, M. Strupler, J.-L. Martin, M-C. Schanne-Klein & E. Beaurepaire. Medecine/Sciences 22, 845-50 (2006). PDF.