Transient Absorption Measurements on Anisotropic Monolayer ReS2.

Anisotropic optical and transport properties of monolayer ReS2 fabricated by mechanical exfoliation are reported. Transient absorption measurements with different polarization configurations and sample orientations reveal that the absorption coefficient and transient absorption are both anisotropic, with maximal and minimal values occurring when the light polarization is parallel and perpendicular to the Re atomic chains, respectively. The maximal values are about a factor of 2.5 of the minimal values. By resolving the spatiotemporal dynamics of excitons, it is found that the diffusion coefficient of excitons moving along Re atomic chains is about 16 cm(2) s(-1) at room temperature, which is about a factor of three larger than those moving perpendicular to that direction. An exciton lifetime of 40 ps is also extracted. These findings establish monolayer ReS2 as an anisotropic 2D transition metal dichalcogenide.

DNA damage may drive nucleosomal reorganization to facilitate damage detection.

One issue in genome maintenance is how DNA repair proteins find lesions at rates that seem to exceed diffusion-limited search rates. We propose a phenomenon where DNA damage induces nucleosomal rearrangements which move lesions to potential rendezvous points in the chromatin structure. These rendezvous points are the dyad and the linker DNA between histones, positions in the chromatin which are more likely to be accessible by repair proteins engaged in a random search. The feasibility of this mechanism is tested by considering the statistical mechanics of DNA containing a single lesion wrapped onto the nucleosome. We consider lesions which make the DNA either more flexible or more rigid by modeling the lesion as either a decrease or an increase in the bending energy. We include this energy in a partition function model of nucleosome breathing. Our results indicate that the steady state for a breathing nucleosome will most likely position the lesion at the dyad or in the linker, depending on the energy of the lesion. A role for DNA binding proteins and chromatin remodelers is suggested based on their ability to alter the mechanical properties of the DNA and DNA-histone binding, respectively. We speculate that these positions around the nucleosome potentially serve as rendezvous points where DNA lesions may be encountered by repair proteins which may be sterically hindered from searching the rest of the nucleosomal DNA. The strength of the repositioning is strongly dependent on the structural details of the DNA lesion and the wrapping and breathing of the nucleosome. A more sophisticated evaluation of this proposed mechanism will require detailed information about breathing dynamics, the structure of partially wrapped nucleosomes, and the structural properties of damaged DNA.

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data.

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.

Detection of structural dynamics by FRET: a photon distribution and fluorescence lifetime analysis of systems with multiple states.

Two complementary methods in confocal single-molecule fluorescence spectroscopy are presented to analyze conformational dynamics by Forster resonance energy transfer (FRET) measurements considering simulated and experimental data. First, an extension of photon distribution analysis (PDA) is applied to characterize conformational exchange between two or more states via global analysis of the shape of FRET peaks for different time bins. PDA accurately predicts the shape of FRET efficiency histograms in the presence of FRET fluctuations, taking into account shot noise and background contributions. Dynamic-PDA quantitatively recovers FRET efficiencies of the interconverting states and relaxation times of dynamics on the time scale of the diffusion time t(d) (typically milliseconds), with a dynamic range of the method of about +/-1 order of magnitude with respect to t(d). Correction procedures are proposed to consider the factors limiting the accuracy of dynamic-PDA, such as brightness variations, shortening of the observation time due to diffusion, and a contribution of multimolecular events. Second, an analysis procedure for multiparameter fluorescence detection is presented, where intensity-derived FRET efficiency is correlated with the fluorescence lifetime of the donor quenched by FRET. If a maximum likelihood estimator is applied to compute a mean fluorescence lifetime of mixed states, one obtains a fluorescence weighted mean lifetime. Thus a mixed state is detected by a characteristic shift of the fluorescence lifetime, which becomes longer than that expected for a single species with the same intensity-derived FRET efficiency. Analysis tools for direct visual inspection of two-dimensional diagrams of FRET efficiency versus donor lifetime are presented for the cases of static and dynamic FRET. Finally these new techniques are compared with fluorescence correlation spectroscopy.

Probability distribution analysis of single-molecule fluorescence anisotropy and resonance energy transfer.

Analysis of anisotropy in single-molecule fluorescence experiments using the probability distribution analysis (PDA) method is presented. The theory of anisotropy-PDA is an extension of the PDA theory recently developed for the analysis of Förster resonance energy transfer (FRET) signals [Antonik, M.; et al. J. Phys. Chem. B 2006, 110, 6970]. The PDA method predicts the shape of anisotropy histograms for any given expected ensemble anisotropy, signal intensity distribution, and background. Further improvements of the PDA theory allow one to work with very low photon numbers, i.e., starting from the level of background signal. Analysis of experimental and simulated data shows that PDA has the major advantage to unambiguously distinguish between shot noise broadening and broadening caused by heterogeneities in the sample. Fitting of experimental histograms yields anisotropy values of individual species, which can be directly compared with those measured in ensemble experiments. Excellent agreement between the ensemble data and the results of PDA demonstrates a good absolute accuracy of the PDA method. The precision in determination of mean values depends mainly on the total number of photons, whereas the ability of PDA to detect the presence of heterogeneities strongly depends on the time window length. In its present form PDA can be also applied to computed fluorescence parameters such as FRET efficiency and scatter-corrected fluorescence anisotropy. Extension of the PDA theory to low photon numbers makes it possible to apply PDA to dynamic systems, for which high time resolution is required. In this way PDA is developed as a sensitive tool to detect biomolecular heterogeneities in space and time.

Fluorescence detection with high time resolution: from optical microscopy to simultaneous force and fluorescence spectroscopy.

Picosecond time-resolution fluorescence signal detection over many hours is possible using the time-correlated single photon counting (TCSPC) technique. Advanced TCSPC with clock oscillator set by the pulsed laser and data analysis provides a tool to investigate processes in single molecules on time scale from picoseconds to seconds. Optical imaging techniques combined with TCSPC allow one to study the spatial distribution of fluorescence properties in solution and on a surface. Mechanical manipulation of a single macromolecule by means of an atomic-force microscope makes it possible to detect fluorescence signal changes as a function of mechanical conformations of a fluorescent dye attached to a single DNA molecule.

Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis.

We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a chi2 near unity. The chi2 surface suggests an ultimate level of precision with FRET of < 1% of the Förster radius. Distributions of FRET signals in donor-acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 A inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the Förster radius are ruled out.

Single-molecule detection and identification of multiple species by multiparameter fluorescence detection.

Two general strategies are introduced to identify and quantify single molecules in dilute solutions by employing a spectroscopic method for data registration and specific burst analysis, denoted multiparameter fluorescence detection (MFD). MFD uses pulsed excitation and time-correlated single-photon counting to simultaneously monitor the evolution of the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time and allows for selection of specific events for subsequent analysis. Using the multiple fluorescence dimensions, we demonstrate a dye labeling scheme of oligonucleotides, by which it is possible to identify and separate 16 different compounds in the mixture via their characteristic pattern by MFD. Such identification procedures and multiplex assays with single-molecule sensitivity may have a great impact on screening of species and events that do not lend themselves so easily to amplification, such as disease-specific proteins and their interactions.

Optical characteristics of atomic force microscopy tips for single-molecule fluorescence applications.

Knowledge of the optical properties of atomic force microscopy (AFM) tips is relevant for the combination of optical and force spectroscopy. The luminescence properties of five commercial AFM tips were characterized using a combination of multiparameter fluorescence detection (MFD) and scanning confocal techniques. These include three Si3N4 tips, one silicon tip, and one high-density carbon (HDC) tip grown on top of a silicon tip. Time-decay histograms of the signal were analyzed to determine the strength of scatter, constant background, and fluorescence in the observed signal. Intensity and anisotropy images with optical resolution down to the diffraction limit were generated. The optical signal recorded from the apex of the Si3N4 tips ranged from 0.7 to 1.9 times the count rates from single Rhodamine 110 molecules under similar illumination conditions. The signal is predominantly composed of scatter and background (>85%), plus a small fluorescence component with lifetimes between 1 and 3 ns. The intensity of the recorded signal fell with increasing distance from the apex, and by 300 nm the signals fell below single-molecule levels for all Si3N4 cantilevers. Silicon cantilevers demonstrated very low count rates relative to single-molecule measurements under all conditions, and virtually no fluorescence. The high-density carbon tips also demonstrated low count rates, but the signal contained a short lifetime fluorescence component (0.7 ns). The intensity of the signals from each of the tips was geometry dependent, demonstrating the highest intensities at the edges and corners. Likewise, the anisotropy of all tip signals was observed to be geometry dependent, with the dependence varying on a case-by-case basis. The implications for using confocal illumination instead of total internal reflection are discussed.