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A bichromophoric model system&sbd;a short peptide labeled with tetramethylrhodamine (TMR) and the carbocyanine Cy5&sbd;embedded in poly(vinyl alcohol) (PVA) was used to investigate energy transfer and collective blinking effects in multichromophoric systems at the level of single molecules. Experiments using direct excitation of the acceptor show evidence of photoinduced reverse intersystem crossing (T1 → TN → S1 → S0) in single Cy5 molecules. We observed that even when the Cy5 fluorophore is in the triplet state, it continues to act as an energy transfer acceptor. This demonstrates that singlet−triplet energy transfer occurs, and can lead to efficient quenching of the fluorescence of the whole bichromophoric system.

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.

Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 HB units have been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg plot (Θ/n vs (n − 2)/n) of the data obtained in solution can be interpreted as an indication for the presence of a chiral secondary structure of the oligoester chain&sbd;on the very short time scale of UV spectroscopy! The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin, rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the distance between the fluorescent groups, thus providing information about the conformation of the OHB chain. With a calculated Förster distance R0 of 60 Å the distance (in 10^-6 M CHCl3) between the termini of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Å, respectively. This result is compatible with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate while the former do not:  The investigation described here did not lead to a decision as to whether an OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 21- or a 31-helical arrangement in solution.