Abstract
The plasma membrane of a cell is an ordered environment, giving rise to anisotropic orientations and restricted motion of constituent lipids and proteins. The membrane environment is also dynamic and heterogeneous, which is important for the regulation of membrane-localized signaling. A number of fluorescent microscopy approaches enable the membrane order to be quantified with high spatial and temporal resolution. A polarization-resolved fluorescence method, termed fluorescent linear dichroism (fLD) imaging, can quantify the orientation of membrane bound fluorophores which allows spatially resolved measurement of membrane order and sub-resolution membrane topology (ruffling). Here we describe the detailed methods for performing fLD imaging in biological membrane environments such as the plasma membrane of living cells. This includes the preparation of the sample with appropriate fluorescent dyes, the requirements of the microscope system, the data collection protocol, and post-acquisition image processing, analysis, and interpretation.
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Acknowledgements
We would like to thank Bjorn Őnfelt (Karolinska Institutet) and Tim Lei (University of Colorado) for critical reading of the manuscript. This work was supported by NIH grant DK085145 (RKPB).
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Benninger, R.K.P. (2015). Fluorescence Linear Dichroism Imaging for Quantifying Membrane Order. In: Owen, D. (eds) Methods in Membrane Lipids. Methods in Molecular Biology, vol 1232. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1752-5_14
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DOI: https://doi.org/10.1007/978-1-4939-1752-5_14
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1751-8
Online ISBN: 978-1-4939-1752-5
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