Single molecule fluorescence measurements provide detailed information about sub-populations, transition intermediates and kinetic data for systems that are not able to be synchronized by removing the ensemble average. One commonly used fluorescence technique is single molecule Foerster Resonance Energy Transfer (smFRET), where precise distance measurements can be performed between the energy transfer efficiency values of ∼10% to ∼90%. These smFRET measurements often need to be performed in solution where Brownian diffusion contributes to non-uniform transits through a laser beam focused into solution. I extend the capability of solution based smFRET with two developments. The first is a characterization of the contribution and propagation of shot noise in the photon counting data collection and analysis. With this understanding, I apply a maximum entropy deconvolution to smFRET histograms to remove the extra broadening due to shot noise to better resolve the experimentally relevant distribution. I also improve upon solution based data collection with the Single Molecule Recycler.
The Single Molecule Recycler is a device that combines a constant drift, by establishing electroosmotic flow, and a confinement in two spatial dimensions, on the order of ∼500 nm each, to make the transits through the laser focus more uniform compared to the Brownian diffusing case. After the fluorescent molecule passes through the focus, a reversing potential changes the direction of the drift velocity and the reversed drift pushes the same molecule back through the laser focus. This process can be repeated hundreds of times for the same molecule and the variable time before reversal can extend the total measurement time from millisecond times up to 15 seconds. With the Single Molecule Recycler it is also possible to perform other single molecule fluorescence measurements. Fluorescence correlation spectroscopy (FCS) can be performed for one molecule to determine the flow speed, hydrodynamic radius or other kinetic data. I could also extract the hydrodynamic radius or the diffusion coefficient from one molecule. An energy transfer efficiency histogram can be created for one molecule in solution and a kinetic time series for one single molecule can be recorded.
|School:||University of Rochester|
|School Location:||United States -- New York|
|Source:||DAI-B 71/06, Dissertation Abstracts International|
|Subjects:||Biomedical engineering, Optics, Biophysics|
|Keywords:||Membrane proteins, Nanochannels, Single molecule recycling|
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