Quantitative imaging of membrane lipid order in cells and organisms

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.

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

“…In particular, analysis of the fluorescence of the di-4-ANEPPDHQ probe, which exhibits an emission shift independent of local chemical composition under different lipid packing conditions (Jin et al, 2005;Demchenko et al, 2009;Dinic et al, 2011), recently enabled the imaging of plant membrane domains at the micrometer scale (Liu et al, 2009). The relevance of this approach has been confirmed by mapping membrane domains using generalized anisotropy-based images of di-4-ANEPPDHQ-stained T cell immunological synapses (Owen et al, 2010c), together with the characterization of membrane organization of nonadherent cells (such as living zebrafish embryo tissues) labeled with this dye (Owen et al, 2012a).…”

“…4A). We used a stringent methodology previously described in the literature for noise removal from fluorescent images (Owen et al, 2012a). This analysis eliminates ROIs that exhibit a fluorescence intensity less than 7% of the highest intensity exhibited by a ROI within the analyzed membrane surface.…”

Nanotubes connecting B lymphocytes: High impact of differentiation-dependent lipid composition on their growth and mechanics

“…This combined effect may undoubtedly enhance the ability of PC to induce positive curvature in the mature B cell membranes. In addition, we found that the membrane level of raft-forming [21,34] disaturated (db=0) PC species almost doubled ( Figure 3C). Furthermore, docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) species were found at significantly elevated amounts in A20 relative to 38C13 cells ( Figure 3D).…”

“…Since dynamic tension and molecular order of PM may also be critical factors in NT growth, the effect of PM hyperfluidization by polyunsaturated fatty acid (PUFA) treatment on NT formation capacity was also investigated. B cells and the connecting NTs were characterized in terms of membrane lipid packing density by imaging di-4-ANEPPHQ polarimetric probe fluorescence and analyzing the generalized polarization [20,21]. Finally, we also examined the mechanical stability of NTs and their sensitivity to environmental mechanical effects/stress.…”

“…Di-4-ANEPPDHQ (Life Technologies) polarimetric fluorescent probe was used to detect lipid order in the B cell membranes or NTs, using a method described previously [21].…”