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Optimization of Cell Morphology Measurement via Single-Molecule Tracking PALM

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  • Nicholas A Frost
  • Hsiangmin E Lu
  • Thomas A Blanpied

Abstract

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.

Suggested Citation

  • Nicholas A Frost & Hsiangmin E Lu & Thomas A Blanpied, 2012. "Optimization of Cell Morphology Measurement via Single-Molecule Tracking PALM," PLOS ONE, Public Library of Science, vol. 7(5), pages 1-10, May.
  • Handle: RePEc:plo:pone00:0036751
    DOI: 10.1371/journal.pone.0036751
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    References listed on IDEAS

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    1. Unknown, 1992. "Table of Contents," 1992 Conference: New Technologies and Innovations in Agricultural Economics Instruction 202951, University of Kentucky, Department of Agricultural Economics.
    2. Unknown, 1992. "Table of Contents," 1992 Occasional Paper Series No. 6 197725, International Association of Agricultural Economists.
    3. Alexandros Pertsinidis & Yunxiang Zhang & Steven Chu, 2010. "Subnanometre single-molecule localization, registration and distance measurements," Nature, Nature, vol. 466(7306), pages 647-651, July.
    4. Marianne Renner & Yegor Domanov & Fanny Sandrin & Ignacio Izeddin & Patricia Bassereau & Antoine Triller, 2011. "Lateral Diffusion on Tubular Membranes: Quantification of Measurements Bias," PLOS ONE, Public Library of Science, vol. 6(9), pages 1-11, September.
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    Cited by:

    1. Julie A Cass & C David Williams & Julie Theriot, 2020. "A Bayesian framework for the detection of diffusive heterogeneity," PLOS ONE, Public Library of Science, vol. 15(5), pages 1-16, May.

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