Single-molecule detection by two-photon-excited fluorescence

We present a technique for observing single fluorophore molecules in solution. A mode-locked laser beam is focused into the solution, thereby defining a two-photon excitation volume localized in three dimensions. Molecules diffusing into and out of this volume produce fluorescence bursts, which are detected with a high signal-to-background ratio. The theoretical foundations for the technique are laid out, including the attendant fluorescence rate distribution, and agree with experimental results obtained for Rhodamine B molecules in water.