Lipid bilayer characterization
Lipid bilayer characterization is the use of various optical, chemical and physical probing methods to study the properties of lipid bilayers. Many of these techniques are elaborate and require expensive equipment because the fundamental nature of the
Fluorescence Microscopy
This potential complication has been given an argument against the use of one of fluorescence recovery after photobleaching (FRAP) to determine bilayer diffusion coefficients. In a typical FRAP experiment a small (~30 µm diameter) area is photobleached by exposure to an intense light source. This area is then monitored over time as the “dead” dye molecules diffuse out and are replaced by intact dye molecules from the surrounding bilayer. By fitting this recovery curve it is possible to calculate the diffusion coefficient of the bilayer.[1][2] An argument against the use of this technique is that what is actually being studied is the diffusion of the dye, not the lipid.[3] While correct, this distinction is not always important, since the mobility of the dye is often dominated by the mobility of the bilayer.
In traditional fluorescence microscopy the resolution has been limited to approximately half the wavelength of the light used. Through the use of
Electrical
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. Accordingly, electrical characterization was one of the first tools used to study the properties of model systems such as black membranes. It was already known that the cell membrane was capable of supporting an ionic gradient and that this gradient is responsible for the ability of
Fundamentally, all electrical measurements of bilayers involve the placement of an electrode on either side of the membrane. By applying a bias across these electrodes and measuring the resulting current, it is possible to determine the
Optical
Lipids are highly
Hydrated bilayers show rich vibrational dynamics and are good media for efficient vibrational energy transfer. Vibrational properties of lipid monolayers and bilayers has been investigated by ultrafast spectroscopic techniques[11] and recently developed computational methods.[12]
AFM
Although AFM is a powerful and versatile tool for studying lipid bilayers, there are some practical limitations and difficulties. Because of the fragile nature of the bilayer, extremely low scanning forces (typically 50pN or less[13][17]) must be used to avoid damage. This consideration is particularly important when studying metastable systems such as vesicles adsorbed on a substrate, since the AFM tip can induce rupture and other structural changes.[18] Care must also be taken to choose an appropriate material and surface preparation for the AFM tip, as hydrophobic surfaces can interact strongly with lipids and disrupt the bilayer structure.[19]
Electron microscopy
In
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
Neutron and X-ray scattering
Both X-rays and high-energy neutrons are used to probe the structure and periodicity of biological structures including bilayers because they can be tuned to interact with matter at the relevant (angstrom-nm) length scales. Often, these two classes of experiment provide complementary information because each has different advantages and disadvantages. X-rays interact only weakly with water, so bulk samples can be probed with relatively easy sample preparation. This is one of the reasons that x-ray scattering was the technique first used to systematically study inter-bilayer spacing.[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.[23] One limitation of x-ray techniques is that x-rays are relatively insensitive to light elements such as hydrogen. This effect is a consequence of the fact that x-rays interact with matter by scattering off of electron density which decreases with decreasing atomic number. In contrast, neutrons scatter off of nuclear density and nuclear magnetic fields so sensitivity does not decrease monotonically with z. This mechanism also provides strong isotopic contrast in some cases, notably between hydrogen and deuterium, allowing researchers to tune the experimental baseline by mixing water and deuterated water. Using reflectometry rather than scattering with neutrons or x-rays allow experimenters to probe supported bilayers or multilayer stacks. These measurements are more complicated to perform an analyze, 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.[25]
References
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