Submillisecond detection of single rhodamine molecules in water

Using a modified confocal fluorescence microscope and a CW argon laser, we have measured fluorescence bursts from diffusing single Rh6G molecules that clearly exceed the background intensity. The exact average number of molecules in the observable volume elements was measured directly via the fluorescence intensity autocorrelation function. This allowed us to estimate the probability of finding several molecules simultaneously in the volume element. A tradeoff between the number of detected fluorescence photons and the signal-to-background ratio was observed. In a volume element of 0.24 fl, 4 photoelectrons on average were detected from a molecule of Rh6G with a fluorescence-to-background ratio of 1000, while the volume element of 60 fl yielded on average 100 photoelectrons with a background of 25 counts. In fast single-molecule detection the intersystem crossing into the triplet state plays an important role, affecting the maximum emission rate from the molecule.