Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution.
An important goal of membrane biology is to define the local heterogeneity of membrane lipid composition. Here we describe a quantitative electron microscopic method that enables the localization of specific membrane lipids at the nanometer scale. The method involves freezing cells rapidly to halt the molecular motion, physically stabilizing membrane molecules in the freeze-fracture replica by the deposition of evaporated platinum and carbon layers and labeling with specific probes for electron microscopic observation. Lipids in both the outer and inner membrane leaflets can thus be labeled, and their distributions can be analyzed quantitatively by statistical methods. A major advantage of this method is that it does not require the expression of artificial probes. Therefore, this method can be applied to any cell in vitro or in vivo, and the whole procedure can be completed in 1-2 d.
Fujita
Akikazu
A
Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Cheng
Jinglei
J
Fujimoto
Toyoshi
T
D000818:Animals; D021941:Caveolae / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D005614:Freeze Fracturing; D066298:In Vitro Techniques; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D015336:Molecular Probe Techniques; D036103:Nanotechnology
D008081:Liposomes; D008563:Membrane Lipids