ANALYSIS OF FLUORESCENCE LIFETIME DATA FOR SINGLE RHODAMINE MOLECULESIN FLOWING SAMPLE STREAMS

Fluorescence events for individual molecules of Rhodamine 110 dye in a stream of methanol occur as bursts of 5-300 photoelectrons when the d ye molecules transit the volume interrogated by the pulsed excitation laser. The arrival times for these events, extracted by use of time-co rrelated single-photon counting techniques, are distributed exponentia lly over a approximately 10-ns time window gated to discriminate again st prompt scattered tight. Still, residual scattered light and solvent -impurity fluorescence contribute significantly to the recorded time d istributions, which therefore must be analyzed as sums of an exponenti al decay and a background. Results obtained from two-parameter nonline ar least-squares fits are compared with maximum-likelihood estimates w hich ignore the background. In agreement with predictions, the relativ e error in the estimated decay rate exceeds the ideal N-1/2 limit that holds for N counts, free of background, recorded over an infinite tim e window. Data sets containing a small number of counts can exhibit pr oblematic behavior, which can be understood from examination of contou r plots of chi2 as a function of the two least-squares parameters. The radiative lifetime of Rhodamine 110 in methanol is estimated to be 4. 2 +/- 0.2 ns.