Abstract
Multi-wavelength single molecule fluorescence microscopy is a valuable tool for clarifying transcription mechanisms, which involve multiple components and intermediates. Here we describe methods for the analysis and interpretation of such single molecule data. The methods described include those for image alignment, drift correction, spot discrimination, as well as robust methods for analyzing single-molecule binding and dissociation kinetics that account for non-specific binding and photobleaching. Finally, we give an example of the use of the resulting data to extract the kinetic mechanism of promoter binding by a bacterial RNA polymerase holoenzyme.