The triplet-state characteristics of the Cy5 molecule related to trans-cis isomerization are investigated by means of ensemble and single molecule measurements. Cy5 has been used frequently in the past 10 years in single molecule spectroscopic applications, e.g., as a probe or fluorescence resonance energy transfer acceptor in large biomolecules. However, the unknown spectral properties of the triplet state and the lack of knowledge on the photoisomerization do not allow us to interpret precisely the unexpected single molecule behaviors. This limits the application of Cy5. The laser photolysis experiments demonstrate that the trans triplet state of Cy5 absorbs about 625 nm, the cis ground state absorbs about 690 nm, and the cis triplet state also absorbs about 690 nm. In other words, the T1-Tn absorptions largely overlap the ground-state absorptions for both trans and cis isomers, respectively. Furthermore, the observation of the cis triplet state indicates an important isomerization pathway from the trans-S1 state to the cis-T1 state upon excitation. The detailed spectra presented in this article let us clearly interpret the exact mechanisms responsible for several important and unexpected photophysical behaviors of single Cy5 molecules such as reverse intersystem crossing (RISC), the observation of dim states with a lower emission intensity and slightly red-shifted fluorescence, and unusual energy transfer from donor molecules to dark Cy5 molecules acting as acceptors in single molecule fluorescence resonance energy transfer (FRET) measurements. Spectral results show that the dim state in the single molecule fluorescence intensity time traces originated from cis-Cy5 because of a lower excitation rate, resulting from the red-shifted ground-state absorption of cis-Cy5 compared to that of the trans-Cy5.

The direct observations of delayed fluorescence and phosphorescence from the cyanine dye Cy5 are reported. The delayed fluorescence is generated from the S(1) state of trans-Cy5 through a reserve intersystem crossing from the cis-triplet state T(1) to the trans-singlet state S(1) via thermal activation. The lowest cis-triplet state is evidenced to be involved in the formation of the isomer. The back-isomerization from cis-triplet state to trans-singlet state crossing, a remarkably back-isomerization pathway that has not been reported before, plays a significant role in this unusual delayed fluorescence.

We present a new class of polymeric dyes bearing the difluoroboraindacene (BODIPY) chromophore within the main chain. Starting from a diiodinated BODIPY monomer, homo-and copolymers with a fully conjugated backbone were efficiently synthesized by transition-metal-catalyzed polycondensation reactions. The photophysical properties of the resulting polymeric materials were investigated in bulk and at the single molecule level. It was found that the BODIPY homopolymer resembles the absorption and emission properties of the individual BODIPY chromophore. In contrast, the copolymer products of 1,4-diethynylbenzene and benzene exhibit absorption and emission spectra that are shifted hypsochromically and bathochromically in regard to the homopolymer, respectively, allowing for easy color tuning by the choice of comonomers. The fluorescence quantum yield of the BODIPY homopolymer is remarkably high (57%). The exceptional brightness of the materials was confirmed in the single molecule investigations; the BODIPY homopolymer emitted several times more photons than the well-established fluorescent probe Rhodamine 6G with a quantum yield close to unity.