Freeze-Fracture Electron Microscopy on Domains in Lipid Mono- and Bilayer on Nano-Resolution Scale
Freeze-fracture electron microscopy (FFEM) as a cryo-fixation, replica, and transmission electron microscopy technique is unique in membrane bilayer and lipid monolayer research because it enables us, to excess and visualize pattern such as domains in the hydrophobic center of lipid bilayer as well as the lipid/gas interface of the lipid monolayer. Since one of the preparatory steps of this technique includes fracturing the frozen sample and, since during this fracturing process the fracture plane follows the area of weakest forces, these areas are exposed allowing us to explore the pattern built up by lipids and/or intrinsic proteins and which are also initiated by peptides, drugs, and toxins reaching into these normally hard to access areas. Furthermore, FFEM as a replica technique is applicable to objects of a large size range and combines detailed imaging of fine structures down to nano-resolution scale within images of larger biological or artificial objects up to several ten’s of micrometers in size.
Biological membranes consist of a multitude of components which can self-organize into rafts or domains within the fluid bilayer characterized by lateral inhomogeneities in chemical composition and/or physical properties. These domains seem to play important roles in signal transduction and membrane traffic. Furthermore, lipid domains are important in health and disease and make an interesting target for pharmacological approaches in cure and prevention of diseases such as Alzheimer, Parkinson, cardiovascular and prion diseases, systemic lupus erythematosus and HIV. As a cryofixation technique FFEM is a very powerful tool to capture such domains in a probe-free mode and explore their dynamics on a nano-resolution scale.
Key wordsFreeze-fracture electron microscopy Domain exploration Liposomal bilayer Lipid monolayer Lipid-stabilized gas bubbles Membranes
The author would like to thank Mr. Stephen Kuzmic, S & J Services, Santa Clara for all his technical support especially in building all the home-made devices, Mr. John Ayou, Microanalytical Laboratories, Inc., Emeryville for excess to the JEOL 100CX, Mr. Alexander Veynberg, UC Berkeley for his excellent workshop-work, and Dr. Jack Ackrell for all the helpful discussions and his technical help especially on preparation days.
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