Single-molecule fluorescence - Each photon counts

The first experiments on single molecules were driven by the challenge of reaching the ultimate limit in chemical analysis. Following the first absorption experiments [1], fluorescence excitation detection facilitated single-molecule spectroscopy with an almost unbelievable signal-to-noise ratio. Fluorescence excitation spectroscopy allowed for the investigation of properties of single molecules and for the comparison of the time average of these properties with the ensemble average. In all of these measurements, the detected photons were integrated to obtain an intensity as a function of one or several experimental parameters in a way similar to ensemble fluorescence spectroscopy. In other words, each photon was just a “click” that increased the number of counts in a certain channel by one.