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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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References

  1. Murphy, D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 40, 325–438 (2001).

    Article  CAS  Google Scholar 

  2. Martin, S. & Parton, R.G. Caveolin, cholesterol, and lipid bodies. Semin. Cell Dev. Biol. 16, 163–174 (2005).

    Article  CAS  Google Scholar 

  3. Murphy, D.J. & Vance, J. Mechanisms of lipid-body formation. Trends Biochem. Sci. 24, 109–115 (1999).

    Article  CAS  Google Scholar 

  4. Imanishi, Y., Gerke, V. & Palczewski, K. Retinosomes: new insights into intracellular managing of hydrophobic substances in lipid bodies. J. Cell Biol. 166, 447–453 (2004).

    Article  CAS  Google Scholar 

  5. Heid, H.W. & Keenan, T.W. Intracellular origin and secretion of milk fat globules. Eur. J. Cell Biol. 84, 245–258 (2005).

    Article  CAS  Google Scholar 

  6. Greenspan, P., Mayer, E.P. & Fowler, S.D. Nile red: a selective fluorescent stain for intracellular lipid droplets. J. Cell Biol. 100, 965–973 (1985).

    Article  CAS  Google Scholar 

  7. Zipfel, W.R., Williams, R.M. & Webb, W.W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003).

    Article  CAS  Google Scholar 

  8. Zipfel, W.R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second-harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7080 (2003).

    Article  CAS  Google Scholar 

  9. Barad, Y., Eisenberg, H., Horowitz, M. & Silberberg, Y. Nonlinear scanning laser microscopy by third harmonic generation. Appl. Phys. Lett. 70, 922–924 (1997).

    Article  CAS  Google Scholar 

  10. Squier, J.A., Müller, M., Brakenhoff, G.J. & Wilson, K.R. Third harmonic generation microscopy. Opt. Express 3, 315–324 (1998).

    Article  CAS  Google Scholar 

  11. Oron, D. et al. Depth-resolved structural imaging by third-harmonic generation microscopy. J. Struct. Biol. 147, 3–11 (2004).

    Article  Google Scholar 

  12. Boyd, R.W. Nonlinear optics 2nd edn. (Academic Press, San Diego, 2003).

    Google Scholar 

  13. Cheng, J.-X. & Xie, X.S. Green's function formulation for third harmonic generation microscopy. J. Opt. Soc. Am. B 19, 1604–1610 (2002).

    Article  CAS  Google Scholar 

  14. Débarre, D., Supatto, W. & Beaurepaire, E. Structure sensitivity in third-harmonic generation microscopy. Opt. Lett. 30, 2134–2136 (2005).

    Article  Google Scholar 

  15. Kajzar, F. & Messier, J. Original technique for third-harmonic-generation measurements in liquids. Rev. Sci. Instr. 58, 2081–2085 (1987).

    Article  CAS  Google Scholar 

  16. Delahunty, T.J. & Rubinstein, D. Accumulation and release of triglycerides by rat liver following partial hepatectomy. J. Lipid Res. 11, 536–543 (1970).

    CAS  PubMed  Google Scholar 

  17. Shteyer, E., Liao, Y., Muglia, L.J., Hruz, P.W. & Rudnick, D.A. Disruption of hepatic adipogenesis is associated with impaired liver regeneration in mice. Hepatology 40, 1322–1332 (2004).

    Article  CAS  Google Scholar 

  18. Welte, M.A., Gross, S.P., Postner, M., Block, S.M. & Wieschaus, E.F. Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics. Cell 92, 547–557 (1998).

    Article  CAS  Google Scholar 

  19. Débarre, D. et al. Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos. Opt. Lett. 29, 2881–2883 (2004).

    Article  Google Scholar 

  20. Sun, C.-K. et al. Higher harmonic generation microscopy for developmental biology. J. Struct. Biol. 147, 19–30 (2004).

    Article  Google Scholar 

  21. Supatto, W. et al. In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses. Proc. Natl. Acad. Sci. USA 102, 1047–1052 (2005).

    Article  CAS  Google Scholar 

  22. Cox, G., Moreno, N. & Feijo, J. Second-harmonic imaging of plant polysaccharides. J. Biomed. Opt. 10, 024013 (2005).

    Article  Google Scholar 

  23. Beaurepaire, E. & Mertz, J. Epifluorescence collection in two-photon microscopy. Appl. Opt. 41, 5376–5382 (2002).

    Article  Google Scholar 

  24. Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J. & Charpak, S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J. Neurosci. Methods 111, 29–37 (2001).

    Article  CAS  Google Scholar 

  25. Dietl, P. & Haller, T. Exocytosis of lung surfactant: from the secretory vesicle to the air-liquid interface. Annu. Rev. Physiol. 67, 595–621 (2005).

    Article  CAS  Google Scholar 

  26. Dirami, G. et al. Lung retinol storing cells synthesize and secrete retinoic acid, an inducer of alveolus formation. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, L249–L256 (2004).

    Article  CAS  Google Scholar 

  27. Huang, S., Heikal, A.A. & Webb, W.W. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys. J. 82, 2811–2825 (2002).

    Article  CAS  Google Scholar 

  28. Patterson, G.H., Knobel, S.M., Arkhammar, P., Thastrup, O. & Piston, D.W. Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H reponses in pancreatic islet beta cells. Proc. Natl. Acad. Sci. USA 97, 5203–5207 (2000).

    Article  CAS  Google Scholar 

  29. Nan, X., Cheng, J.-X. & Xie, X.S. Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy. J. Lipid Res. 44, 2202–2208 (2003).

    Article  CAS  Google Scholar 

  30. Niemz, M.H. Laser-Tissue Interactions: fundamentals and Applications (Springer, Berlin, 2004).

    Google Scholar 

  31. Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 μm in living brains by use of a TiAl2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    Article  CAS  Google Scholar 

  32. Beaurepaire, E., Oheim, M. & Mertz, J. Ultra-deep two-photon excitation in turbid media. Opt. Commun. 188, 25–29 (2001).

    Article  CAS  Google Scholar 

  33. Day, C.P. & James, O.F.W. Hepatic steatosis: Innocent bystander or guilty party? Hepatology 27, 1463–1466 (1998).

    Article  CAS  Google Scholar 

  34. Bosshard, C., Gubler, U., Kaatz, P., Mazerant, W. & Meir, U. Non-phase-matched optical third-harmonic generation in noncentrosymmetric media: cascaded second-order contributions for the calibration of third-order nonlinearities. Phys. Rev. B 61, 10688–10701 (2000).

    Article  CAS  Google Scholar 

  35. Wohlfarth, C. & Wohlfarth, B. Landolt-Börnstein Numerical data and functional relationships in science and technology (New Series III/38A et B) (Springer-Verlag, Berlin, 1996).

    Google Scholar 

  36. Nikogosyan, N.D. Properties of Optical and Laser-related materials: a handbook (Wiley, Chichester, 1997).

    Google Scholar 

Download references

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

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  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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