 Total Internal Reflectance Fluorescence Microscopy (TIRFM) is a technique that allows the investigator to selectively excite fluorophores within 150nm of a glass surface. This will eliminate background fluorescence due to free fluorophore in solution. To implement TIRF, a laser is passed through a prism or a high numerical aperture objective, hitting 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. |