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Visual Discrimination of Membrane Domains in Live Cells by Widefield Microscopy

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Fluorescent Methods to Study Biological Membranes

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 13))

Abstract

Membrane dynamics is a fast-evolving field with the many new methods and probes being developed each year affording ever increased insights into how membranes behave in the laboratory. Typically, these developments are first tested in model membranes using high-cost, bespoke microscopes which often employ confocal and two-photon systems and which give little consideration to preservation of cellular integrity and homeostasis during experiments. This chapter addresses the clear need to rapidly apply and deploy this work into mainstream biological laboratories by development of economical, four-dimensional imaging on user-friendly low-cost systems using widefield optics and simultaneous capture of multiple fluorescent markers. Such systems are enabling biologists to consider the coordinated processes triggered from signalling platforms during cellular interaction with the environment. In this chapter, we describe the progress made to date and in particular we focus on the Laurdan family of fluorescent probes, which are being used to image whole cells and tissues using widefield epifluorescence microscopy and which can be usefully combined with simultaneous capture at longer wavelengths (yellow through far red) for imaging of cell morphology or for following expressed markers such as fluorescent adaptor proteins.

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Acknowledgements

We thank Dr. Paul Thomas at The Wellcome Laboratory for cell imaging for critical reading of the manuscript and advice and insight along the way and Rosanna Davies for her tremendous illustrative skills in producing the drawing of our widefield Laurdan microscopy setup.

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Authors and Affiliations

  1. Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, England

    Claire E. Butler, Guy Wheeler & Kevin M. Tyler

  2. Cairn Research Ltd, Graveney Road, Faversham Kent, ME13 8UP, UK

    Jeremy Graham

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  1. Claire E. Butler
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  2. Guy Wheeler
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  3. Jeremy Graham
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  4. Kevin M. Tyler
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Correspondence to Kevin M. Tyler .

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Editors and Affiliations

  1. Faculté de Pharmacie, Laboratoire de Biophotonique, Université de Strasbourg, route du Rhin 74, Illkirch, 67401, France

    Yves Mély

  2. Faculté de Pharmacie, Laboratoire de Biophotonique et, Université de Strasbourg, route du Rhin 74, Illkirch, 67401, France

    Guy Duportail

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Butler, C.E., Wheeler, G., Graham, J., Tyler, K.M. (2012). Visual Discrimination of Membrane Domains in Live Cells by Widefield Microscopy. In: Mély, Y., Duportail, G. (eds) Fluorescent Methods to Study Biological Membranes. Springer Series on Fluorescence, vol 13. Springer, Berlin, Heidelberg. https://doi.org/10.1007/4243_2012_47

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