Single-molecule detection with a two-photon fluorescence microscope with fast-scanning capabilities and polarization sensitivity.
We describe a laser-scanning two-photon fluorescence microscope that is capable of observing single molecules with excellent temporal resolution and three-dimensional spatial resolution. To demonstrate the capabilities of the instrument we present single-molecule fluorescence data obtained in several different scanning modes. In addition, a polarization-sensitive detection scheme can provide detailed three-dimensional information about the orientations of molecules in any of these scanning modes.