Lipid bilayer characterization
Over the past two decades, a new generation of characterization tools including AFM has allowed the direct probing and imaging of membranes in situ with little to no chemical or physical modification.
More recently, dual polarisation interferometry has been used to measure the optical birefringence of lipid bilayers to characterise order and disruption associated with interactions or environmental effects.
This is a particularly contentious issue when studying the diffusion or phase separation of lipids, as both processes are very sensitive to the size and shape of the molecules involved.
By studying the destructive interference patterns formed it is possible to individually resolve the two leaflets of a supported bilayer and determine the distribution of a fluorescent dye in each.
[5] Electrical measurements are the most straightforward way to characterize one of the more important functions of a bilayer, namely its ability to segregate and prevent the flow of ions in solution.
This resistance is typically quite high for intact bilayers, often exceeding 100 GΩ since the hydrophobic core is impermeable to charged hydrated species.
The most common system is the silver/silver chloride electrode since this reaction is stable, reversible, involves a single electron transfer and can produce large currents.
[9] In addition to simple DC current measurements it is also possible to perform AC electrical characterization to extract information about the capacitance and complex impedance of a bilayer.
This effect, studied by dual polarisation interferometry has been used to measure dynamic reorganisation of the layer due to temperature, ionic strength, and molecular interactions with e.g. antimicrobial peptides.
AFM is a promising technique because it has the potential to image with nanometer resolution at room temperature and even underwater, conditions necessary for natural bilayer behavior.
[13] Another AFM experiment performed in a tapping mode under aqueous buffer medium allowed (1) to determine the formation of transmembrane pores (holes) around nanoparticles of approximately 1.2 to 22 nm diameter via subtraction of AFM images from series recorded during the lipid bilayer formation and (2) to observe adsorption of single insulin molecules onto exposed nanoparticles.
[14] Another advantage is that AFM does not require fluorescent or isotopic labeling of the lipids, as the probe tip interacts mechanically with the bilayer surface.
[20] 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.
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.)
[22] X-ray scattering can also yield information on the average spacing between individual lipid molecules, which has led to its use in characterizing phase transitions.
These measurements are more complicated to perform an analysis, but allow determination of cross sectional composition, including the location and concentration of water within the bilayer.
[24] In the case of both neutron and x-ray scattering measurements, the information provided is an ensemble average of the system and is therefore subject to uncertainty based on thermal fluctuations in these highly mobile structures.
