-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Applied Spectroscopy 09/2011; 65(9):981-90. · 1.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
The Journal of Physical Chemistry B 06/2010; 114(23):7983-95. · 3.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
The Journal of Physical Chemistry B 09/2007; 111(34):10253-62. · 3.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Microscopy Research and Technique 06/2007; 70(5):433-41. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc.
Microscopy Research and Technique 03/2007; 70(5):433 - 441. · 1.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
The Journal of Physical Chemistry B 04/2006; 110(13):6970-8. · 3.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Analytical Chemistry 04/2006; 78(6):2039-50. · 5.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
ChemPhysChem 06/2005; 6(5):976-83. · 3.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid- binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Angstrom shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.
Proceedings of the National Academy of Sciences 03/2003; · 9.68 Impact Factor
