Third harmonic generation (THG) microscopy

Third harmonic generation (THG) microscopy
THG imaging of a live zebrafish larva. Adapted from Ferrer Ortas et al, Light Sci App 2023.

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

In particular, third-harmonic generation (THG) microscopy highlights sub-micrometer interfaces (e.g. lipidic structures) and provides 3D structural images of unstained samples. Polarized THG can also detect optical anisotropy. At specific wavelengths, resonant THG highlights red blood cells. This information can be combined with other nonlinear signals such as SHG, fluorescence (2PEF, 3PEF) or fluorescence lifetime (FLIM).

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 susceptibility), and we develop novel applications such as :
• imaging tissue morphology;
• using THG to detect myelin, lipids or hemoglobin;
• using polarized THG to probe organized media: biocrystals, cornea, etc

Related publications

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy
J. Ferrer Ortas, P. Mahou, S. Escot, C. Stringari, N. B. David, L. Bally-Cuif, N. Dray, M. Négrerie, W. Supatto, E. Beaurepaire
Light: Science & Applications (2023).
Kidney stone classification using multimodal multiphoton microscopy
M. Gleeson, J. Morizet, P. Mahou, M. Daudon, D. Bazin, C. Stringari, M.-C. Schanne-Klein, E. Beaurepaire
ACS Photonics (2023).
Third harmonic imaging contrast from tubular structures in the presence of index discontinuity
J. Morizet, N. Olivier, P. Mahou, A. Boutillon, C. Stringari, E. Beaurepaire
Scientific Reports (2023).
Modeling nonlinear microscopy near index-mismatched interfaces
J. Morizet, G. Sartorello, N. Dray, C. Stringari, E. Beaurepaire, N. Olivier
Optica (2021).
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 (2019)
THG imaging and analysis of the effect of low gravity on the lacuno-canalicular network of mouse bone
R. Genthial, M. Gerbaix, D.Farlay, L. Vico, E. Beaurepaire, D. Débarre, A. Gourrier
PLoS ONE (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, E. Beaurepaire
Opt. Express 20(22), 24886-24902 (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, E. Beaurepaire
Biomed. Opt. Express 2(10), 2837-2849 (2011).
Advances in multiphoton microscopy for imaging embryos
W. Supatto, T.V. Truong, D. Debarre, 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).
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).
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).