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  • Open Access

Probing Ordered Lipid Assemblies with Polarized Third-Harmonic-Generation Microscopy

Maxwell Zimmerley, Pierre Mahou, Delphine Débarre*, Marie-Claire Schanne-Klein, and Emmanuel Beaurepaire

  • Laboratory for Optics and Biosciences, École Polytechnique, CNRS UMR 7645, INSERM U696, 91128 Palaiseau, France

  • *Present address: Laboratory of Interdisciplinary Physics, Université Joseph Fourier, CNRS UMR 5588, 38402 Saint Martin d’Hères, France
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Phys. Rev. X 3, 011002 – Published 14 January, 2013

DOI: https://doi.org/10.1103/PhysRevX.3.011002

Abstract

Ordered lipid assemblies are responsible for important physiological functions including skin barrier and axon conductivity. However, techniques commonly used to probe molecular order such as X-ray scattering and nuclear magnetic resonance are not suited for in-situ tissue studies. Here, we identify and characterize a novel contrast mechanism in nonlinear optical microscopy which is sensitive to molecular ordering in multilamellar lipid vesicles (MLVs) and in samples obtained from human skin biopsy: polarized third-harmonic generation (P-THG). We develop a multiscale theoretical framework to calculate the anisotropic, nonlinear optical response of lipid arrays as a function of molecular order. This analysis reveals that conserved carbon-carbon bond and aliphatic tail directionality are the atomic- and molecular-scale sources of the observed P-THG response, respectively. Agreement between calculations and experiments on lipid droplets and MLVs validates the use of P-THG as a probe of lipid ordering. Finally, we show that P-THG can be used to map molecular ordering in the multilamellar, intercorneocyte lipid matrix of the stratum corneum of human skin. These results provide the foundation for the use of P-THG in probing molecular order and highlight a novel biomedical application of multiphoton microscopy in an optically accessible tissue relevant to monitoring lipid-related disorder.

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Popular Summary

Bilayer structures formed by ordered assemblies of the chemically “bigamous” lipid molecules (which have chemical affinity for both water and oil) are building blocks found in all mammalian biological cells. In particular, the proper formation of stacked lipid bilayers plays a key role in the function of skin as a protective barrier, and in the electrical connectivity of neurons. Probing and measuring the lipid-molecular ordering in biological tissues, or in situ, and correlating that knowledge with the functions of the tissues is therefore scientifically, and even medically, important. In this study, we establish a novel method for characterizing in-situ lipid ordering in human skin samples with subcellular 3D resolution. We do so by exploiting and refining a nonlinear (multiphoton) microscopy technique that depends on third-harmonic generation and on light polarization.

Ordered lipid structures are in fact nonlinear optical materials. When illuminated by an optical (laser) beam, they interact with the incident photons and emit photons with multiples of the energy of the original photons. Third-harmonic generation corresponds to the photon emission of tripled energy or frequency and is the basis of our method. As we have discovered through a theoretical analysis that spans from the molecular scale to the lipid-assembly scale, the light resulting from third-harmonic generation in a lipid-bilayer assembly is actually sensitive to how the molecules (or more specifically the chemical bonds in the molecules) are oriented with respect to the polarization of the excitation beam. Since ordering of lipid molecules in a supramolecular assembly is intimately related to their orientations in the assembly, the observable sensitivity to molecular orientation translates into an observable sensitivity to the molecular ordering. Based on this theoretical understanding, we are able to experimentally image, with micrometer resolution, lipid orientation and order not only in artificial lipid assemblies, but also in tissue samples obtained from human skin biopsy. Compared to the existing techniques for characterizing lipid order, such as nuclear magnetic resonance, the optical method represents a huge improvement in spatial resolution, can be used in situ, and has the advantage of not requiring the use of unnatural bulky probes.

Our proof-of-principle study demonstrates the promise of polarization-sensitive third-harmonic-generation microscopy for probing molecular order in biological tissues. We hope that further development of the method, by us and by others, will turn it into a practical method for physiological studies of ordered molecular assemblies.

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Phys. Rev. X 3, 011002– Published 14 January, 2013
  • Received 4 August 2012
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This article is available under the terms of the Creative Commons Attribution 3.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.

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