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Identification of Intensity Ratio Break Points from Photon Arrival Trajectories in Ratiometric Single Molecule Spectroscopy
Department of Chemistry, Williams College, 47 Lab Campus Drive, Williamstown, MA 01267, USA
San Francisco Estuary Institute, Oakland, CA 94621, USA
* Author to whom correspondence should be addressed.
Received: 19 April 2012; in revised form: 7 June 2012 / Accepted: 12 June 2012 / Published: 18 June 2012
Abstract: We describe a statistical method to analyze dual-channel photon arrival trajectories from single molecule spectroscopy model-free to identify break points in the intensity ratio. Photons are binned with a short bin size to calculate the logarithm of the intensity ratio for each bin. Stochastic photon counting noise leads to a near-normal distribution of this logarithm and the standard student t-test is used to find statistically significant changes in this quantity. In stochastic simulations we determine the significance threshold for the t-test’s p-value at a given level of confidence.We test the method’s sensitivity and accuracy indicating that the analysis reliably locates break points with significant changes in the intensity ratio with little or no error in realistic trajectories with large numbers of small change points, while still identifying a large fraction of the frequent break points with small intensity changes. Based on these results we present an approach to estimate confidence intervals for the identified break point locations and recommend a bin size to choose for the analysis. The method proves powerful and reliable in the analysis of simulated and actual data of single molecule reorientation in a glassy matrix.
Keywords: single molecule spectroscopy; ratiometric detection; statistical analysis; glass dynamics
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Bingemann, D.; Allen, R.M. Identification of Intensity Ratio Break Points from Photon Arrival Trajectories in Ratiometric Single Molecule Spectroscopy. Int. J. Mol. Sci. 2012, 13, 7445-7465.
Bingemann D, Allen RM. Identification of Intensity Ratio Break Points from Photon Arrival Trajectories in Ratiometric Single Molecule Spectroscopy. International Journal of Molecular Sciences. 2012; 13(6):7445-7465.
Bingemann, Dieter; Allen, Rachel M. 2012. "Identification of Intensity Ratio Break Points from Photon Arrival Trajectories in Ratiometric Single Molecule Spectroscopy." Int. J. Mol. Sci. 13, no. 6: 7445-7465.