T he current renaissance of the use of the fluorescence resonance energy-transfer (FRET) (1) process between two weakly coupled dipoles, one acting as donor and the other acting as acceptor, results from various elegant and successful studies of distance changes in individual biomolecules by using singlemolecule spectroscopy (SMS). This type of study is often referred to as single-pair Förster resonance ET (2-4). Recent advances in fluctuation microscopy allow the observation of changes in the ET efficiency in single-pair Förster resonance ET systems at the millisecond-to-nanosecond time scale (5, 6). It has been suggested that, in the latter time scale, thermally excited conformational dynamics of (bio)macromolecules will become accessible (5). Thus far, FRET measurements at these time scales have overlooked competitive Förster-type ET pathways, which might complicate data analysis and interpretation of the FRET data. The FRET process involves nonradiative transfer from a donor to an acceptor. For molecules within the weak coupling limit, Förster derived an expression for the rate constant of dipole-dipole-induced ET (7). This relation shows that the ET efficiency scales with the sixth power of the distance between the chromophores:Here, R 0 is the distance at which the efficiency equals 50%, i.e., the distance at which an equal probability exists for the excited chromophore to relax to the ground state via emission of a photon or to undergo ET. It includes the spectral properties and the relative orientation (in terms of the orientation factor 2 ) of donor and acceptor transition dipoles. If the distances and orientations of the chromophores are kept constant, the ET efficiency is determined by the spectral overlap of the corresponding transitions, i.e., the overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor. Therefore, FRET can also occur between identical molecules if the Stokes shift is small enough to allow for a sufficient overlap of the emission and absorption spectrum of the chromophores. This so-called homo-transfer or energy hopping represents a key mechanism for energy transport in some light-harvesting complexes (8).However, Förster-type resonance ET is not restricted to the nonradiative transfer of energy from a donor in the excited state to a ground-state acceptor. Transfer processes that are allowed within the Förster formalism are those for which there are no changes in electron spin in the acceptor transition. Therefore, even ET processes between donor and acceptor molecules with different spin multiplicity likewise are possible. Hence, the transfer of excitation energy from a chromophore residing in the first excited singlet (S) state to another chromophore residing either in the triplet (T) or in an excited singlet state are possible and competitive ET pathways.In the present contribution, we demonstrate that, because of the specific excitation conditions that are applied in SMS, indeed several Förster-type ET pathways are prevalent in single bichromopho...

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.