We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET. Fluorescence resonance energy transfer (FRET) has found wide use in structural biology, biochemistry, and polymer science for measuring distances in the 10-to 80-A range (1-5).In FRET, energy is transferred from a donor molecule to an acceptor molecule via an induced-dipole, induced-dipole interaction, with the transfer efficiency E depending on the inverse-sixth-power of the distance R between the donor and acceptor: E = 1/(1+[R/R0]6), where Ro is the distance at which 50% of the energy is transferred. Ro is a function of the properties of the dyes and the relative orientation of their dipole moments: R. = [8.79 x 10-5 J(A) OD n-4 K2]116 [A]. J(A) is the spectral overlap of donor emission and acceptor absorption [nm4_M-1.cm-1], OD is the donor quantum yield, n is the index of refraction of the medium, and K2 is a geometrical factor which accounts for the relative orientation of the two dipoles.Near-field scanning optical microscopy (NSOM) (6-8) is a relatively new technique that allows optical measurements with sub-wavelength resolution. It is based on a probe consisting of a very small (sub-wavelength) aperture that is placed in close proximity (in the near field; <10 nm) to the sample under study. By using the probe as an excitation source, fluorescence of a single molecule has been detected (9). The emission spectra (10) and excited state lifetime (11-13) of a single molecule have also been measured. Another important aspect of near-field detection is that the optical radiation in the near field has an electric field component along its direction of propagation (in contrast to far-field radiation). This allows mapping the transition dipole moment orientation of a single fluorescent molecule in three dimensions (9).The marriage between FRET and NSOM offers many potential advantages when distance and orientation information is required on a molecular level. Here we list several. (i) Because the orientation of donor and acceptor can potentially be measured, the uncertainty in K2, which is often a large source of uncertainty i...

We observed and made unambiguous distinctions between abrupt photophysical events of single molecules: a rotational jump of a single dipole, a transition to a dark state (reversible and irreversible photobleaching), and a spectral jump. The study was performed in the far field by modulating the excitation polarization and monitoring the fluorescence in time. This technique also allowed us to measure the in-plane dipole orientation of stationary single molecular dipoles with subdegree accuracy and to resolve desorption and readsorption of fluorophores from and onto a glass surface. In one case, clear evidence was obtained for rapid rotation of the dipole after a desorption process.

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