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Quantitative imaging of membrane lipid order in cells and organisms

Nature Protocols volume 7, pages 24–35 (2012)Cite this article

Abstract

It is now recognized that lipids and proteins in cellular membranes are not homogenously distributed. A high degree of membrane order is the biophysical hallmark of cholesterol-enriched lipid rafts, which may induce the lateral sorting of proteins within the membrane. Here we describe a quantitative fluorescence microscopy technique for imaging localized lipid environments and measuring membrane lipid order in live and fixed cells, as well as in intact tissues. The method is based on the spectral ratiometric imaging of the polarity-sensitive membrane dyes Laurdan and di-4-ANEPPDHQ. Laurdan typically requires multiphoton excitation, making it suitable for the imaging of tissues such as whole, living zebrafish embryos, whereas di-4-ANEPPDHQ imaging can be achieved with standard confocal microscopes. This approach, which takes around 4 h, directly examines the organization of cellular membranes and is distinct from alternative approaches that infer membrane order by measuring probe partitioning or dynamics.

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Figure 1: Schematic of the probes and their emission spectra in different lipid environments.
Figure 2: Initialization of lasers for Laurdan microscopy in the Leica LAS software.
Figure 3: Activation of lasers for Laurdan microscopy in the Leica LAS software.
Figure 4: Detection settings for Laurdan microscopy in the Leica LAS software.
Figure 5: Scan settings for Laurdan microscopy in the Leica LAS software.
Figure 6: Generating GP images and pseudocolored GP-intensity-merged images.
Figure 7: Laurdan or di-4-ANEPPDHQ GP image processing.
Figure 8: Laurdan imaging of live zebrafish embryos.
Figure 9: Laurdan imaging of live HeLa cells.
Figure 10: Di-4-ANEPPDHQ imaging of live HeLa cells.

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Acknowledgements

D.M.O. and K.G. acknowledge funding from the Australian Research Council; K.G. also receives funding from the National Health and Medical Research Council of Australia and the Human Frontier Science Program. C.R. is grateful to the CONSOLIDER-INGENIO fellowship (CSD2009-00016, Ministerio de Innovación, Ciencia y Tecnología).

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Authors and Affiliations

  1. Centre for Vascular Research, University of New South Wales, Sydney, Australia

    Dylan M Owen, Astrid Magenau, Ahmed Abu-Siniyeh & Katharina Gaus

  2. Facultat de Medicina, Departament de Biologia Cel.lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain

    Carles Rentero

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Contributions

D.M.O. developed the di-4-ANEPPDHQ protocol and wrote the manuscript. C.R. wrote the GP analysis macro. D.M.O. and A.M. conducted the experiments presented here. D.M.O., A.M. and A.A-S. developed the zebrafish protocol. K.G. developed the Laurdan protocol and wrote the manuscript.

Corresponding authors

Correspondence to Dylan M Owen or Katharina Gaus.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

ImageJ macro for processing two channel Laurdan or di-4-ANEPPDHQ into GP images. The macro imports TIFF images from two different spectral windows. Using the data flow shown in Figure 6, it generates GP images and pseudo-coloured GP-intensity merged images. (TXT 16 kb)

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Owen, D., Rentero, C., Magenau, A. et al. Quantitative imaging of membrane lipid order in cells and organisms. Nat Protoc 7, 24–35 (2012). https://doi.org/10.1038/nprot.2011.419

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