by Andre on 25 December 2005
…How I strongly suspect that you’re a single fluorescently labeled protein. I sometimes think I’m doing astronomy instead of biophysics when I see the type of data we collect. The movie shown here is a sequence of TIRFM images from single titin molecules adsorbed on a surface. TIRFM, short for total internal reflection fluorescence microscopy, is a technique that takes advantage of some neat physics to observe biology at its finest scales. Let me explain.
When light hits an interface between two materials, in our case glass and water, under certain conditions it will be entirely reflected at that interface. One way to figure out these conditions is by solving Maxwell’s equations for a propagating wave and applying the appropriate boundary conditions. Part of the result is Snell’s Law, which you could also derive using Fermat’s interesting principle. Either way, you’ll find that when the light is incident on the interface from the higher refractive index side, there is some critical angle above which no light propagates through the interface. Now for the neat physics. Although there is no propagating light on the other side of the interface, the electric field is not zero: there’s an exponentially decaying evanescent field and it’s this field that we use to excite the fluorophores.
One of the key features of this field is that it decays very quickly. The result is that only fluorophores within a few hundred nanometers of the surface get excited thus reducing the unwanted fluorescence from the rest of the sample. In fact, the background is cut down sufficiently to observe the fluorescence from individual molecules on the surface. The principle and some applications are outlined on many microscope manufacturers’ websites like Olympus, Nikon, and Zeiss. You can also find some image galleries with some impressive multicoloured shots of cells’ otherwise private parts.