Total internal reflectance fluorescence microscopy (TIRFM) is a technique that allows the investigator to selectively excite fluorophores on or near a surface. This will eliminate background fluorescence due to free fluorophore in solution. To implement TIRF, a laser is passed through a prism so as to hit a glass: liquid interface at an angle greater than the critical angle. Under this condition the light will totally internally reflect rather than refract through the interface, setting up an evanescent field at the interface, which decays exponentially away into the liquid. This decay creates an effective layer of light (i.e. ~150 nm) that will excite only those fluorophores at the interface surface or in the thin layer of liquid. This technique combined with high sensitivity imaging systems allows one to detect the emission from a single fluorophore.
We have recently
combined TIRFM with the laser trap
assay. Our goal is to
record real time molecular structure and functional
data from a single myosin molecule as it interacts
with actin. In addition, with the use of
fluorescently labeled ATP real time correlation of
myosin's molecular mechanics and enzymology will be
possible.
