Skip to main content Accessibility help

Video-Based Tracking of Single Molecules Exhibiting Directed In-Frame Motion

  • M. Yavuz Yüce (a1), Alexandr Jonáš (a1), Alper Kiraz (a1) and Alper T. Erdoğan (a2)


Trajectories of individual molecules moving within complex environments such as cell cytoplasm and membranes or semiflexible polymer networks provide invaluable information on the organization and dynamics of these systems. However, when such trajectories are obtained from a sequence of microscopy images, they can be distorted due to the fact that the tracked molecule exhibits appreciable directed motion during the single-frame acquisition. We propose a new model of image formation for mobile molecules that takes the linear in-frame motion into account and develop an algorithm based on the maximum likelihood approach for retrieving the position and velocity of the molecules from single-frame data. The position and velocity information obtained from individual frames are further fed into a Kalman filter for interframe tracking of molecules that allows prediction of the trajectory of the molecule and further improves the precision of the position and velocity estimates. We evaluate the performance of our algorithm by calculations of the Cramer-Rao Lower Bound, simulations, and model experiments with a piezo-stage. We demonstrate tracking of molecules moving as fast as 7 pixels/frame (12.6 μm/s) within a mean error of 0.42 pixel (37.43 nm).


Corresponding author

Corresponding author. E-mail:


Hide All
Abraham, A.V., Ram, S., Chao, J., Ward, E.S. & Ober, R.J. (2009). Quantitative study of single molecule location estimation techniques. Opt Express 17, 2335223373.
Abraham, A.V., Ram, S., Chao, J., Ward, E.S. & Ober, R.J. (2010). Comparison of estimation algorithms in single-molecule localization. Proceedings of SPIE, vol. 7570, 757004-1.
Anthony, S.M. & Granick, S. (2009). Image analysis with rapid and accurate two-dimensional Gaussian fitting. Langmuir 25, 81528160.
Cheezum, M.K., Walker, W.F. & Guilford, W.H. (2001). Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys J 81, 23782388.
Eldar, Y.C. (2008). Rethinking biased estimation: Improving maximum likelihood and the Cramer-Rao bound. Found Trends Signal Process 1, 305449.
Evans, E. (2001). Probing the relation between force—lifetime—and chemistry in single molecular bonds. Annu Rev Biophys Biomol Struct 30, 105128.
Genovesio, A. & Olivo-Marin, J.-C. (2003). Tracking fluorescent spots in biological video microscopy. Proceedings of SPIE, vol. 4964, pp. 98105.
Hamamatsu. (2009). EM-CCD Technical Note. Technical report. Hamamatsu Photonics K.K., Systems Division.
Ho, W. (2002). Single-molecule chemistry. J Chem Phys 117, 1103311061.
Hoffmann, A., Nettels, D., Clark, J., Borgia, A., Radford, S.E., Clarke, J. & Schuler, B. (2011). Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: Recurrence analysis of single particles (RASP). Phys Chem Chem Phys 13, 18571871.
Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. (2008). Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77, 5176.
Kailath, T., Sayed, A.H. & Hassibi, B. (2000). Linear Estimation. Upper Saddle River, NJ: Prentice Hall.
Kay, S.M. (1993). Fundamentals of Statistical Signal Processing, Volume 1: Estimation Theory. Upper Saddle River, NJ: Pearson Education.
Kues, T., Peters, R. & Kubitscheck, U. (2001). Visualization and tracking of single protein molecules in the cell nucleus. Biophys J 80, 29542967.
Moerner, W.E. (2007). New directions in single-molecule imaging and analysis. Proc Nat Acad Sci USA 104, 1259612602.
Ober, R.J., Ram, S. & Ward, E.S. (2004). Localization accuracy in single-molecule microscopy. Biophys J 86, 11851200.
Pampaloni, F., Lattanzi, G., Jonas, A., Surrey, T., Frey, E. & Florin, E.L. (2006). Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length. Proc Nat Acad Sci USA 103, 1024810253.
Pralle, A., Keller, P., Florin, E.L., Simons, K. & Horber, J.K.H. (2000). Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J Cell Biol 148, 9971007.
Saxton, M.J. & Jacobson, K. (1997). Single-particle tracking: Applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26, 373399.
Schmidt, T., Schutz, G.J., Gruber, H.J. & Schindler, H. (1996). Local stoichiometries determined by counting individual molecules. Anal Chem 68, 43974401.
Schuster, J., Cichos, F. & Borczyskowski, C. (2002). Diffusion measurements by single-molecule spot-size analysis. J Phys Chem A 106, 54035406.
Schutz, G.J., Sonnleitner, M., Hinterdorfer, P. & Schindler, H. (2000). Single molecule microscopy of biomembranes. Mol Membr Biol 17, 1729.
Smal, I., Niessen, W. & Meijering, E. (2008). A new detection scheme for multiple object tracking in florescence microscopy by joint probabilistic data association filtering. Int Symp Biomed Imag: From Nano to Macro (ISBI-08), Paris, France. pp. 264267.
Smith, C.S., Joseph, N., Rieger, B. & Lidke, K.A. (2010). Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7, 373375.
Thompson, R.E., Larson, D.R. & Webb, W.W. (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82, 27752783.
Usman, M., Hero, A., Fessler, J. & Rogers, W. (1993). Bias-variance tradeoffs analysis using uniform CR bound for a SPECT system. Nuclear Science Symposium and Medical Imaging Conference, 1993 IEEE Conference Record. vol. 3, pp. 1463–1467.
Wong, Y., Lin, Z.P. & Ober, R.J. (2011). Limit of the accuracy of parameter estimation for moving single molecules imaged by fluorescence microscopy. IEEE Trans Signal Process 59, 895911.
Wu, P.H., Agarwal, A., Hess, H., Khargonekar, P.P. & Tseng, Y. (2010). Analysis of video-based microscopic particle trajectories using Kalman filtering. Biophys J 98, 28222830.
Yorulmaz, M., Kiraz, A. & Demirel, A.L. (2009). Motion of single terrylene molecules in confined channels of poly(butadiene)-poly(ethylene oxide) diblock copolymer. J Phys Chem B 113, 96409643.


Related content

Powered by UNSILO
Type Description Title
Supplementary materials

Yüce Supplementary Material
Supplementary Material 1

 PDF (62 KB)
62 KB
Supplementary materials

Yüce Supplementary Material
Supplementary Material 2

 PDF (645 KB)
645 KB

Video-Based Tracking of Single Molecules Exhibiting Directed In-Frame Motion

  • M. Yavuz Yüce (a1), Alexandr Jonáš (a1), Alper Kiraz (a1) and Alper T. Erdoğan (a2)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.