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Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy

Nature Methods volume 3, pages 47–53 (2006)Cite this article

Abstract

Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1–2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with ∼1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues.

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Figure 1: Lipid bodies are the main source of contrast in THG images of hepatocytes and liver tissue.
Figure 2: Lipid bodies trafficking dynamics can be studied in gastrulating D. melanogaster embryos using THG microscopy.
Figure 3: THG imaging of plant seed tissue.
Figure 4: Epi-detected imaging is possible in weakly absorbing tissues owing to THG light backscattering.
Figure 5: Multimodal multiphoton imaging of fresh lung tissue.

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Acknowledgements

We thank M. Welte (Brandeis University, Waltham, Massachusetts, USA) for advice on embryo staining, G. Ephritikhine (Institut des Sciences du Végétal, Gif-sur-Yvette, France) for help with the plant experiments, J.-M. Sintes and X. Solinas for assistance in microscope design, S. Boucherie for assistance in hepatocytes preparation, and J.-L. Martin and J. Ogilvie for critical comments. This work was supported by the Délégation Générale pour l'Armement.

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Authors and Affiliations

  1. Laboratory for optics and biosciences, Ecole Polytechnique, CNRS, INSERM, Palaiseau, F-91128, France

    Delphine Débarre, Ana-Maria Pena, Marie-Claire Schanne-Klein & Emmanuel Beaurepaire

  2. Laboratory of physico-chemistry of life, CNRS, Institut Curie, Paris, F-75005, France

    Willy Supatto

  3. Pathophysiology and epidemiology of respiratory failure, INSERM, Bichat Hospital, Paris, F-75018, France

    Aurélie Fabre

  4. Laboratory of cell signaling and calcium, INSERM, Paris XI University, Orsay, F-91405, France

    Thierry Tordjmann & Laurent Combettes

Authors
  1. Delphine Débarre
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Correspondence to Emmanuel Beaurepaire.

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Supplementary information

Supplementary Figure 1

THG imaging of isolated hepatocytes in different physiological states. (PDF 46 kb)

Supplementary Figure 2

THG-2PEF imaging of plant seed tissue labeled with Nile Red. (PDF 170 kb)

Supplementary Figure 3

Fluorescence emission spectrum of lipid bodies in lung tissue. (PDF 13 kb)

Supplementary Video 1

Simultaneous THG(purple)-2PEF(red) 3D imaging of a hepatocyte stained with Nile Red, a hydrophobic fluorescent dye that accumulates in lipid bodies. (MOV 1866 kb)

Supplementary Video 2

THG image sequence showing the trafficking dynamics of lipid bodies in a live D. melanogaster embryo during cellularization. (MOV 2149 kb)

Supplementary Video 3

Individual lipid body tracked using THG microscopy in a developing D. melanogaster embryo. (MOV 1393 kb)

Supplementary Data

Cell viability after THG imaging. (PDF 297 kb)

Supplementary Methods (PDF 105 kb)

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Débarre, D., Supatto, W., Pena, AM. et al. Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy. Nat Methods 3, 47–53 (2006). https://doi.org/10.1038/nmeth813

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