single molecule ( sm ) techniques are relatively new additions to the field of biophysics that allow one to manipulate individual molecules and study their behavior . to make these studies more relevant to what actually happens in the cell 
 , one needs to move beyond the studies of individual molecules in isolation and study many different molecules working in concert . 
 this presents a technical challenge as most sm experiments measure only one observable as a function of time , whereas complex biomolecular systems require multidimensional sm analysis 
 . frster resonance energy transfer ( fret ) is one of the most common single molecule approaches and can report on the real time distance changes . 
 however , fret requires two fluorophores which will ultimately limit the degree of multiplexing in future sm applications 
 . it will be useful if a single fluorophore can be used to provide equivalent information . in this communication 
 , we show that fluorescence lifetime analysis of a single cy3 fluorophore attached to the promoter region of the dna can be used to reveal transient reaction intermediates during transcription initiation by t7 rna polymerase . 
 this work represents the first demonstration of real - time biochemical reactions observed via single molecule fluorescence lifetime trajectories of immobilized molecules .