Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

Abstract

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...

NSOM Investigations of the Spectroscopy and Morphology of Self-Assembled Multilayered Thin Films

“…As mentioned above, fluorescence resonance energy transfer can be used to measure donor/acceptor distances in the 10-80 Å range. 32 The energy-transfer efficiency E depends on the inverse-sixth-power of the distance R between the donor and the acceptor, as follows R 0 is the distance at which 50% of the energy is transferred from the donor to the acceptor.…”

Quantifying molecular colocalization in live cell fluorescence microscopy

“…The right image resembles a reconstruction of the left image, where areas of high colocalization can directly be classified. [1][2][3][4][5][6][7][8][9][10][11][12]. In FRET molecular interaction partners, such as proteins, antibodies, or short DNA or RNA strands, are labeled with donor and acceptor fluorophores that need to be in close proximity (1-10 nm) to each other to ensure the efficient transfer of excitation energy.…”

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

“…The first demonstration of spFRET from immobilized molecules at room temperature was performed using a near-field microscope [83]. Since then, many groups have applied spFRET to study biomolecules and biomolecular complexes using microscopies (such as confocal or EFFM) that are easily adapted to an aqueous environment.…”