Publications

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The objectives of this project were to develop a new scientific tool for studies of chemical processes at the single molecule level, and to provide enhanced capabilities for multiplexed, ultrasensitive separations and immunoassays. We have combined microfluidic separation techniques with our newly developed technology for spectrally and temporally resolved detection of single molecules. The detection of individual molecules can reveal fluctuations in molecular conformations, which are obscured in ensemble measurements, and allows detailed studies of reaction kinetics such as ligand or antibody binding. Detection near the single molecule level also enables the use of correlation techniques to extract information, such as diffusion rates, from the fluorescence signal. The micro-fluidic technology offers unprecedented control of the chemical environment and flow conditions, and affords the unique opportunity to study biomolecules without immobilization. For analytical separations, the fluorescence lifetime and spectral resolution of the detection makes it possible to use multiple parameters for identification of separation products to improve the certainty of identification. We have successfully developed a system that can measure fluorescence spectra, lifetimes and diffusion constants of the components of mixtures separated in a microfluidic electrophoresis chip.

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Over the past few years we have developed the ability to acquire images through a confocal microscope that contain, for each pixel, the simultaneous fluorescence lifetime and spectra of multiple fluorophores within that pixel. We have demonstrated that our system has the sensitivity to make these measurements on single molecules. The spectra and lifetimes of fluorophores bound to complex molecules contain a wealth of information on the conformational dynamics and local chemical environments of the molecules. However, the detailed record of spectral and temporal information our system provides from fluorophores in single molecules has not been previously available. Therefore, we have studied several fluorophores and simple fluorophore-molecule systems that are representative of the use of fluorophores in biological systems. Experiments include studies of a simple fluorescence resonance energy transfer (FRET) system, green fluorescent probe variants and quantum dots. This work is intended to provide a basis for understanding how fluorophores report on the chemistry of more complex biological molecules.

Single molecule fluorophores were studied for the first time with a new confocal fluorescence microscope that allows the wavelength and emission time to be simultaneously measured with single molecule sensitivity. In this apparatus, the photons collected from the sample are imaged through a dispersive optical system onto a time and position sensitive detector. This detector records the wavelength and emission time of each detected photon relative to an excitation laser pulse. A histogram of many events for any selected spatial region or time interval can generate a full fluorescence spectrum and correlated decay plot for the given selection. At the single molecule level, this approach makes entirely new types of temporal and spectral correlation spectroscopy of possible. This report presents the results of simultaneous time- and frequency-resolved fluorescence measurements of single rhodamine 6G (R6G), tetramethylrhodamine (TMR), and Cy3 embedded in thin films of polymethylmethacrylate (PMMA).

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