Biomedical subjects

Matthew Antonik

Publications and source records attributed to Matthew Antonik.

3 recordsLinked to original sources

PubMed2006

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.

DNA↗

PubMed2006

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.

Fluorescence Polarization↗

PubMed2005

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.

Anisotropy↗