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.
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Farrer et al. (1999) studied this question.
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