Improving z-tracking accuracy in the two-photon single-particle tracking microscope

TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) microscope. This method utilizes a maximum likelihood estimator (MLE) to determine the particle's 3D position that maximizes the likelihood of the observed time-correlated photon count distribution. Our Monte Carlo simulations show that the MLE-based tracking scheme can improve the z-tracking accuracy of TSUNAMI microscope by 1.7 fold. In addition; MLE is also found to reduce the temporal correlation of the z-tracking error. Taking advantage of the stnaller and less temporally correlated z-tracking error, we have precisely recovered the hybridization-melting kinetics of a DNA model system from thousands of short single-particle trajectories in silica. Our method can be generally applied to other 3D single-particle tracking techniques. (C) 2015 AIP Publishing LLC.