1 A Road Map to Single Molecule Dynamics

Signals from single fluorescent dye molecules were first measured in non-biological environments. Some of the earliest studies acquired signals from singlefluorescence dyes embedded in a solid matrix at the temperature of liquid helium [1] and from excitation fluorescence signals [2]. In early studies carried out at room temperature, single fluorescence dye molecules spread over a film were observed using near-field scanning fluorescence microscopy [3]. In a biological environment where fluorescence is largely quenched by the surrounding water molecules, single molecule imaging was first accomplished in 1995 using total internal reflected fluorescence (TIRF) and epi-fluorescence micros-copy [4] (see Chapter 2). Single fluorophores attached to myosin molecules were visualized in aqueous solution in the vicinity of a glass surface while single ATP turnover from the samemyosin molecule was measured using fluorescently-labeled ATP. TIRF microscopy has been widely used for single molecule imaging. Other studies have measured single fluorophores bound to biomolecules in solution while passing through a fixed small volume using confocal microscopy [5]