Imaging individual molecules by two-photon excitation

Wide-field epi-illumination of supported lipid membranes by a femtosecond near-infrared laser allowed us to identify and follow individual fluorescence labeled lipids in their motion within the membrane. The fluorophores were excited by two-photon absorption of the near-infrared beam as seen from a quadratic dependence of the fluorescence signal on the illumination intensity. The largely decreased background signal on two-photon excitation compared to that using one-photon excitation suggests that single-molecule imaging on native cell membranes will come into reach.