Lipid Bilayer Characterization - Fluorescence Microscopy

Fluorescence Microscopy

Fluorescence microscopy is a technique whereby certain molecules can be excited with one wavelength of light and will emit another longer wavelength of light. Because each fluorescent molecule has a unique spectrum of absorption and emission, the location of particular types of molecules can be determined. Natural lipids do not fluoresce, so it is always necessary to include a dye molecule in order to study lipid bilayers with fluorescence microscopy. To some extent, the addition of the dye molecule always changes the system, and in some cases it can be difficult to say whether the observed effect is due to the lipids, the dye or, most commonly, some combination of the two. The dye is usually attached either to a lipid or a molecule that closely resembles a lipid, but since the dye domain is relatively large it can alter the behavior of this other molecule. 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.

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. An argument against the use of this technique is that what is actually being studied is the diffusion of the dye, not the lipid. 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 confocal microscopy and image processing this limit can be extended, but typically not much below 100 nanometers, which is much smaller than a typical cell but much larger than the thickness of a lipid bilayer. More recently, advanced microscopy methods have allowed much greater resolution under certain circumstances, even down to sub-nm. One of the first of these methods to be developed was Förster resonance energy transfer (FRET). In FRET, two dye molecules are chosen such that the emission spectrum of one overlaps the absorption spectrum of the other. This energy transfer is extremely distance dependent, so it is possible to tell with angstrom resolution how far apart the two dyes are. This can be used for instance to determine when two bilayers fuse and their components mix. Another high resolution microscopy technique is fluorescence interference contrast microscopy (FLIC). This method requires that the sample be mounted on a precisely micromachined reflective surface. 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.