The single molecule fluorescence spectroscopy of various isolated single-chromophoric dye molecules and multiple-chromophoric conjugated polymer molecules has been investigated. For each system the transient fluorescence “intensity”, I CW(t) (i.e., detected photons/dwell time), has been recorded with continuous wave (CW) irradiation. I CW(t) has been analyzed to yield an occurrence histogram for the different “intensities”, H(I), and an intensity time-autocorrelation function C I (t). The histograms H(I) for the various examples show highly diverse behavior with one, two, or even three peaks as well as “flat regions”. The different features in the histograms are shown to arise from distinct photophysical processes. From the study of model systems, characteristic features in the intensity histograms and autocorrelation functions are shown to result from photon shot noise, “blinking” due to triplet bottlenecks, spectral diffusion due to environmental fluctuations, and interchromophoric energy transfer. Classification of the relevant photophysical processes is aided by single molecule spectroscopic data on these systems, including wavelength-resolved emission spectroscopy and “two-color excitation spectroscopy”, as well as stochastic simulations. The results indicate that a combined analysis of H(I) and C I (t) is a valuable approach in sorting out single molecule behavior involving multiple photophysical processes in complex systems. For single molecule systems that exhibit “on−off blinking” involving the formation of dark states, the paper also explores the practical advantages of studying the duration histograms (H(t on) and H(t off)) versus the intensity autocorrelation function C I (t), for quantifying the underlying photophysical dynamics.

ton. To collect the remaning sufonics ~ndividually, we then renlected the sufoncs n t o the C , which was equpped ~511th a 10 m by 100 lnln Phenomenex (Torrance. CA) star-ion anon exchange column, and eluted them twth a 5.4 m M Na,CO, + 5.1 mM NaHCO, buffer, l?le separated ESA f r o~n a small amount o4 co-eutng sulfate by reinjecting the ti~!o compounds and eluting them with a 3.2 m M Na,CO, + 3 1 NaHCO, m M buffer. Basene separation between MSA and ESNsulfate vias -2 min; betvieen ESA. sulfate and i?SA, t vias -9 min, and between ESA and sulfate, it vias -1 min. Ater re-~noval of the carbonate buffer with HCI, each compound twas then placed n a quanz tube and dred by rotary evaporation (maxmu~n temperature -50'C).Copper oxde was then placed :n each tube, and h:gh vacuum, with m~lo warmin9 (<10S3C), was used for co~npete drying. The sa~nples v/ere then sealed and combusted to sulfate, CO,, an0 H, O at 500'C. The CO, and H 2 0 were collected w~t h a proced~re smiar to that of Epsten et a1 (73). The H, O vias con\!ened to H by the method of Cole~nan and Moore (27) viith a znc catalyze f r o~n J. M. Hayes of Indiana Unversit),. The CO, and H were then analyzed w~t h 6-60-Nuclide and 3-60-HD-Nuclide mass spectrometers. The sulfate vias extracted from each tube with hydrochorc acid an0 prepared for S isotopic analysis as in (6).

We report here the direct measurement of intra-tRNA distances during annealing of the tRNA primer to the HIV RNA genome. This key step in the initiation of retroviral reverse transcription involves hybridization of one strand of the acceptor arm of a specific lysine tRNA to the primer binding site on the RNA genome. Although the mechanism of tRNA unwinding and annealing is not known, previous studies have shown that HIV nucleocapsid protein (NC) greatly accelerates primer/template binary complex formation in vitro. An open question is whether NC alone unwinds the primer or whether unwinding by NC requires the RNA genome. We monitored the annealing process in solution by using f luorescence resonance energy transfer (FRET). Distance measurements demonstrate unequivocally that the tRNA acceptor stem is not substantially unwound by NC in the absence of the RNA genome, that is, unwinding is not separable from hybridization. Moreover, FRET measurements show that both heat-and NC-mediated annealing result in an Ϸ40-Å increase in the separation of the two ends of the tRNA acceptor arm on binding to the template. This large increase in separation of the two ends suggests a complete displacement of the nonhybridized strand of the acceptor stem in the initiation complex.

Van der Waals dimers between benzofuran, dibenzofuran, and fluorene were studied in a supersonic jet. These show red-shifted excimer/exciplex emission following excitation to their locally excited states. The dibenzofuran dimer forms an excimer from its zero-point level, while the dibenzofuran−benzofuran and fluorene−benzofuran dimers require a moderate amount of excess vibrational energy for excimer formation. Exciplex formation in dibenzofuran−fluorene was inferred from spectral broadening. Excimer/exciplex formation was attributed to strong mixing of a charge transfer state with the exciton state. The charge transfer state has a low enough energy at its equilibrium geometry to mix, but this requires a large change in geometry from the equilibrium geometry of the locally excited state. There is a barrier in the adiabatic potential surface between the minima of the locally excited state and the excimer/exciplex state. This barrier is larger for greater differences in equilibrium geometry.

The dependence of the rate of singlet excitation transfer on the donor−acceptor energy gap was investigated in bichromophoric spiranes with symmetry-forbidden zero-order electronic coupling. The fluorescence measurements were performed in a supersonic jet in order to avoid collisional and inhomogeneous line broadening. Fluorescence excitation spectra and single-vibronic-level emission spectra of the model chromophores cyclopentaphenanthrene and 1,8-dimethylnaphthalene and the bichromophores spirofluorenephenanthrene and spirofluorenenaphthalene are presented and analyzed. Although the transition moments of the linked chromophores are rigorously perpendicular and the exchange coupling between the v‘ = 0 states is computationally shown to be zero, all spiranes with energy gaps larger than ∼1000 cm-1 exhibited complete electronic energy transfer from all vibrational states of the electronically excited donor, including the undistorted v‘ = 0 state. This behavior is explained in terms of vibronic coupling between the sparse states of the donor and the dense manifold (pseudocontinuum) of the acceptor states. The electronic energy transfer was sufficiently fast to result in measurable lifetime broadening of the donor absorption lines, from which the k EET was estimated. The results demonstrate that the zero-order picture overestimates the degree of the molecular orbital symmetry control over electronic energy transfer and charge-transfer rates and that at sufficiently high driving forces the vibronically mediated “symmetry-forbidden” electronic energy transfer can be very rapid (∼1 × 1012 s-1).

Fluorescence-on sensors typically rely on disrupting photoinduced electron transfer quenching of the excited state through binding the electron donor. To provide a more general fluorescence-on signaling unit, a quencher-fluorophore dyad has been developed in which quenching by electron transfer to a tethered viologen acceptor can be disrupted through complexation of the viologen by cucurbit[7]uril (CB7). Dyads of benzyl viologen-rhodamine B or a BODIPY fluorophore gave upon CB7 complexation 14- and 30-fold fluorescence enhancement, respectively.