Lipid Bilayer Characterization - Electron Microscopy

Electron Microscopy

In electron microscopy a beam of focused electrons interacts with the sample rather than a beam of light as in traditional microscopy. Electrons have a much shorter wavelength than light so electron microscopy has much higher resolution than light microscopy, potentially down to the atomic scale. Because lipid bilayers are arranged on the molecular level, this higher resolution has been invaluable. In 1960, when the structure of the bilayer was still debated, it was electron microscopy that offered the first direct visualization of the two apposing leaflets. In conjunction with rapid freezing techniques, electron microscopy has also been used to study the mechanisms of inter- and intracellular transport, for instance in demonstrating that exocytotic vesicles are the means of chemical release at synapses. Often, electron microscopy is the only probe technique with sufficient resolution to determine complex nanometer-scale morphologies.

The limitations of electron microscopy in the study of lipid structures deal primarily with sample preparation. Most electron microscopes require the sample to be under vacuum, which is incompatible with hydration at room temperature. To surmount this problem, samples can be imaged under cryogenic conditions with the associated water frozen, or a metallic negative can be made from a frozen sample. It is also typically necessary to stain the bilayer with a heavy metal compound such as osmium tetroxide or uranyl acetate because the low atomic weight constituents of lipids (carbon, nitrogen, phosphorus, etc.) offer little contrast compared to water. If a Transmission electron microscope (TEM) is being used, it is also necessary to cut or polish the sample into a very thin (<1 micrometre) sheet, which can be difficult and time-consuming. Scanning Electron Microscopy (SEM) does not require this step, but cannot offer the same resolution as TEM. Both methods are surface-sensitive techniques and cannot reveal information about deeply buried structures.