Robust Measurement of Membrane Bending Moduli Using Light Sheet Fluorescence Imaging of Vesicle Fluctuations

The mechanical rigidity of lipid membranes is a key determinant of the energetics of cellular membrane deformation. Measurements of membrane bending moduli remain rare, however, and show a large variance, a situation that can be addressed by the development of improved techniques and by comparisons...

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Bibliographic Details
Published in:Langmuir 2013-11, Vol.29 (47), p.14588-14594
Main Authors: Loftus, Andrew F, Noreng, Sigrid, Hsieh, Vivian L, Parthasarathy, Raghuveer
Format: Article
Language:English
Subjects:
Chemistry
Colloidal state and disperse state
Exact sciences and technology
Fluorescence
General and physical chemistry
Humans
Membranes
Microscopy, Fluorescence - instrumentation
Monomeric GTP-Binding Proteins - chemistry
Particle Size
Phosphatidylcholines - chemistry
Surface Properties
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Summary:The mechanical rigidity of lipid membranes is a key determinant of the energetics of cellular membrane deformation. Measurements of membrane bending moduli remain rare, however, and show a large variance, a situation that can be addressed by the development of improved techniques and by comparisons between disparate techniques applied to the same systems. We introduce here the use of selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) to image thermal fluctuations of giant vesicles. The optical sectioning of SPIM enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For both lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, which lowers membrane rigidity in a concentration-dependent manner, we show that the resulting bending modulus values are in close agreement with those derived from an independent assay based on membrane tether pulling. We also show that the fluctuation spectra of vesicles bound by the mammalian Sar1A protein, which stiffens membranes at high concentrations, are not well fit by a model of homogeneous quasi-spherical vesicles, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties induced by protein assemblies.
ISSN:0743-7463
1520-5827
DOI:10.1021/la403837d