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Microscopy Reference Library

Molecular Localization Accuracy in Superresolution Microscopy

In single-molecule superresolution microscopy, individual molecules that are densely packed within the resolution limit (as defined by the point-spread function) can be isolated from one another on the basis of one or more distinguishing optical characteristics. Each molecule can then be localized to a much higher precision (in effect, breaking the diffraction barrier) by determining its center of fluorescence emission through a statistical fit of the ideal point-spread function to its measured photon distribution. In this manner, single-molecule localization to nearly 1-nanometer precision can be readily demonstrated.

Thompson, R. E., Larson, D. R. and Webb, W. W.

Precise nanometer localization analysis for individual fluorescent probes.  Biophysical Journal 82: 2775-2783 (2002).  Perhaps the most cited paper in single-molecule superresolution microscopy, Dr. Webb and co-workers thoroughly review various aspects of calculating the centroid for images of individual fluorescent particles and molecules. Discussed are the factors that limit the precision of these techniques, and a simple equation is derived that describes the precision of localization over a wide range of conditions.

Cheezum, M. K., Walker, W. F. and Guilford, W. H.

Quantitative comparison of algorithms for tracking single fluorescent particles.  Biophysical Journal 81: 2378-2388 (2001).  In this report, the authors compare specific implementations of four commonly used tracking algorithms for single particle tracking. The techniques include cross-correlation, sum-absolute difference, centroid, and direct Gaussian fit.

Yildiz, A., Forkey, J. N., McKinney, S. A., Ha, T., Goldman, Y. E. and Selvin, P. R.

Myosin V walks hand-over-hand: Single fluorophores imaging with 1.5-nm localization.  Science 300: 2061-2065 (2003).  An elegant demonstration of single-molecule localization precision to monitor the progression of fluorescently labeled myosin molecular motors along actin filaments. The authors gain evidence for the hand-over-hand model rather than the inchworm model.

Qu, X., Wu, D., Mets, L. and Scherer, N. F.

Nanometer-localized multiple single-molecule fluorescence microscopy.  Proceedings of the National Academy of Sciences (USA) 101: 11298-11303 (2004).  The authors introduce nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by applying both centroid localization and photobleaching of single fluorophores attached to short duplex strands of DNA.

Churchman, L. S., Okten, Z., Rock, R. S., Dawson, J. F. and Spudich, J. A.

Single molecule high resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.  Proceedings of the National Academy of Sciences (USA) 102: 1419-1423 (2005).  Dr. Churchman and associates present a technique called single-molecule high resolution colocalization (SHREC) of fluorescent dyes that enables the measurement of inter-fluorophore distances in macromolecules with better than 10-nanometer resolution.

Babroff, N.

Position measurement with a resolution and noise-limited instrument.  Review of Scientific Instruments 57:1152-1157 (1986).  An analysis of maximum likelihood estimation of localization accuracy in noise-limited scientific instruments, including optical microscopes. The author demonstrates that the limiting error in position measurement is a simple function of the instrument resolution, the density of data points, and the signal-to-noise ratio of the data.

Ober, R. J., Ram, S. and Ward, E. S.

Localization accuracy in single-molecule microscopy.  Biophysical Journal 86: 1185-1200 (2004).  The authors examine application of the Fisher information matrix to determine the limit of the localization accuracy for a single molecule, which is a function of the emission wavelength, the objective numerical aperture, the optical system efficiency, and the photon emission rate.

Cronin, B., de Wet, B. and Wallace, M. I.

Lucky imaging: Improved localization accuracy for single molecule imaging.  Biophysical Journal 96: 2912-2917 (2009).  By applying an astronomical data-analysis technique known as "Lucky" imaging, the authors show that by selectively discarding data points from individual single molecule trajectories, imaging resolution can be improved by a factor of 1.6 for single fluorophores and up to 5.6 for more complex images.

Gordon, M. P., Ha, T. and Selvin, P. R.

Single-molecule high-resolution imaging with photobleaching.  Proceedings of the National Academy of Sciences (USA) 101: 6462-6465 (2004).  A creative approach to localization of single molecules by using sequential photobleaching behavior to differentiate between individual fluorophores that are too closely spaced to be resolved. The authors demonstrate 5-nanometer localization precision.

Small, A. R.

Theoretical limits on errors and acquisition rates in localizing switchable fluorophores.  Biophysical Journal 96: L16-L18 (2009).  Dr. Small introduces formalism for defining error rates in localizing switchable fluorophores that includes the general relationship between error rates, image acquisition speed, and the performance characteristics of the image processing algorithms.