Alan Van Orden

Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate.

We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species:  Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.

A maximum likelihood estimator to distinguish single molecules by their fluorescence decays

Abstract We have developed a maximum likelihood estimator to distinguish between similar molecules at the single molecule level based upon fluorescence decay measurements. Time resolved fluorescence measurements for single Rhodamine 6G and tetramethylrhodamine isothiocyanate molecules in fluid flow are derived from time-correlated single photon counting. A maximum likelihood estimator is developed and applied to data from a mixture of molecules. Single molecules are identified and distinguished by their fluorescence time decays. Comparison is made between identification error rates and theoretical predictions. To our knowledge, this is the first reported example of single molecule identification by fluorescence decay in a mixture.

Application of Single Molecule Detection to Dna Sequencing

Abstract A flow cytometric, single molecule approach to DNA sequencing is described. A single, fluorescently labeled DNA fragment is suspended in a flow stream. An exonuclease is added to sequentially cleave the end base into the flow stream where it is detected and identified by laser-induced fluorescence.

Millisecond time-scale folding and unfolding of DNA hairpins using rapid-mixing stopped-flow kinetics.

We report stopped-flow kinetics experiments to study the folding and unfolding of 5 base-pair stem and 21 nucleotide polythymidine loop DNA hairpins over various concentrations of NaCl. The reactions occurred on a time scale of milliseconds, considerably longer than the microsecond time scale suggested by previous kinetics studies of similar-sized hairpins. In comparison to a recent fluorescence correlation spectroscopy study (J. Am. Chem. Soc. 2006, 128, 1240-1249), we suggest the microsecond time-scale reactions are due to intermediate states and the millisecond time-scale reactions reported here are due to the formation of the fully folded DNA hairpin. These results support our view that DNA hairpin folding occurs via a minimum three-state mechanism.

Cy3 in AOT reverse micelles I. Dimer formation revealed through steady-state and time-resolved spectroscopy.

Cyanine-3 (Cy3) fluorescent dye molecules confined in sodium di-2-ethylhexyl sulfosuccinate (AOT) reverse micelles were examined using steady-state absorption and emission as well as time-resolved fluorescence spectroscopy to understand the effect of confinement on the spectroscopic properties of the dye. This study explored a wide range of reverse micelle sizes, with hydrodynamic radii ranging from ∼1.7 to ∼5 nm. The relative concentrations of Cy3 and AOT reverse micelles were such that, on average, one dye molecule was present for every 2 × 10(4) to 9 × 10(5) reverse micelles. In the smallest reverse micelles examined, observed changes in the absorption and emission spectra and fluorescence lifetime of the dye molecules indicated H-aggregation of Cy3 into side-by-side dimers. It is hypothesized that this dimerization is governed by the high local concentrations that result from the confinement of the Cy3 in the reverse micelles. What is notable about this study is that this dimer occurs even at overall dye concentrations in the nanomolar range. Such concentrations are too low for aggregation to occur in bulk solution. Hence, the reverse micelles serve as nanocatalysts for this aggregation process.

Probing the ionic atmosphere of single-stranded DNA using continuous flow capillary electrophoresis and fluorescence correlation spectroscopy.

Two-beam fluorescence cross-correlation spectroscopy coupled with continuous flow capillary electrophoresis (2bFCCS-CFCE) was used to study the relationship between diffusion and effective charge of a fluorescently labeled 40-base polythymine single-stranded DNA (ssDNA) as a function of Mg2+ concentration. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the diffusion and electrophoretic migration of ssDNA at a range of Mg2+ concentrations and electric field strengths. The effective charge of the ssDNA could then be determined using simple calculations. It was found that as the Mg2+ concentration in the buffer solution increased, the diffusion of the ssDNA also increased, while the effective charge of the ssDNA decreased. This was believed to be caused by increased association of the Mg2+ counterions with the negatively charged backbone of the ssDNA, which partially neutralized the negatively charged functional groups and allowed the ssDNA to adopt a more compact structure. To our knowledge, this is the first demonstration of the measurement of effective charge of ssDNA in relation to Mg2+ concentration.

RESTRICTED LATERAL DIFFUSION OF LUTEINIZING HORMONE RECEPTORS IN MEMBRANE MICRODOMAINS

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.

Counterion and polythymidine loop-length-dependent folding and thermodynamic stability of DNA hairpins reveal the unusual counterion-dependent stability of tetraloop hairpins.

Stem-loop DNA hairpins containing a 5-base-pair (bp) stem and single-stranded polythymidine loop were investigated using thermodynamic melting analysis and stopped-flow kinetics. These studies revealed the thermodynamic stability and folding kinetics as a function of loop length and counterion concentration. Our results show the unusually high thermodynamic stability for tetraloop or 4 poly(dT) loop hairpin as compared with longer loop length hairpins. Furthermore, this exceptional stability is highly counterion-dependent. For example, in the higher counterion concentration regime of 50 mM NaCl and above, the tetraloop hairpin displays enhanced stability as compared with longer loop length hairpins. However, at lower counterion concentration of 25 mM NaCl and below, the thermal stability of tetraloop hairpin is consistent with the longer loop hairpins. The enhanced stability of tetraloop hairpins at higher counterion concentration can be explained on the basis of the combined entropic effect of loop closure as well as base stacking in the loop regions. The stability of longer loop length hairpins at all counterion concentrations as well as tetraloop hairpin at lower counterion concentration can be explained on the basis of entropic effect of loop closure alone. The thermodynamic parameters at lower and higher counterion concentrations were determined to quantify the enhanced stability of base-stacking effects occurring at higher counterion concentrations. For example, for 100 mM NaCl, excess Gibbs energy and enthalpy due to base stacking within the tetraloops were measured to be -1.2 ± 0.14 and -3.28 ± 0.32 kcal/mol, respectively, whereas, no excess of Gibbs energy and enthalpy was observed for 0, 5, 10, and 25 mM NaCl. These findings suggest significant base-stacking interactions occurring in the loop region of the tetraloop hairpins at higher counterion concentration and less significant base-stacking interactions in the lower counterion concentration regime. We suggest that at higher counterion concentrations, hydrophobic collapse of the nucleotides in the loop may be enhanced due to the increased polarity of the solvent, thereby enhancing base-stacking interactions that contribute to unusually high stability.

Photon Antibunching in Small Clusters of CdSe/ZnS Core/Shell Quantum Dots.

Coincident photon histogram measurements of fluorescence antibunching via confocal microscopy correlated with atomic force microscopy were carried out on (i) individual CdSe/ZnS core/shell quantum dots (QDs), (ii) several well separated QDs, and (iii) clusters of QDs. Individual QDs and well separated QDs showed the expected degree of antibunching for a single emitter and several independent emitters, respectively. The degree of antibunching in small, compact clusters was more characteristic of a single emitter than multiple emitters. The antibunching in clusters provides strong evidence of nonradiative energy transfer between QDs in a cluster. A minimal phenomenological model of energy transfer gives reasonable quantitative agreement with the experimental results.

Correction to “Artifact-Free and Detection-Profile-Independent Higher-Order Fluorescence Correlation Spectroscopy for Microsecond-Resolved Kinetics. 2. Mixtures and Reactions”

Fluorescence correlation spectroscopy (FCS) is a primary tool in the time-resolved analysis of nonreacting or reacting molecules in solution, based on fluorescence intensity fluctuations. However, conventional FCS alone is insufficient for a complete determination of reaction or mixture parameters. In an accompanying article, a technique for the computation of artifact-free higher-order correlations with microsecond time resolution was described. Here, we demonstrate the applications of the technique to analyze the systems of fast and slow reactions. As an example of non- or slow-reacting systems, the technique is applied to resolve two-component mixtures of labeled oligonucleotides. Next, the protonation reaction of fluorescein isothiocyanate in phosphate buffer is analyzed as an example of fast reactions (relaxation time <1 μs). By reference to an (apparent) nonreacting system, the simple factorized form of cumulant-based higher-order correlations is exploited to remove the dependence on the molecular d...