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Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements

Author

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  • Carla Coltharp
  • Rene P Kessler
  • Jie Xiao

Abstract

Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (tThresh and dThresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.

Suggested Citation

  • Carla Coltharp & Rene P Kessler & Jie Xiao, 2012. "Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements," PLOS ONE, Public Library of Science, vol. 7(12), pages 1-15, December.
  • Handle: RePEc:plo:pone00:0051725
    DOI: 10.1371/journal.pone.0051725
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    References listed on IDEAS

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    1. Sarah L Veatch & Benjamin B Machta & Sarah A Shelby & Ethan N Chiang & David A Holowka & Barbara A Baird, 2012. "Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting," PLOS ONE, Public Library of Science, vol. 7(2), pages 1-13, February.
    2. J. Gower & P. Legendre, 1986. "Metric and Euclidean properties of dissimilarity coefficients," Journal of Classification, Springer;The Classification Society, vol. 3(1), pages 5-48, March.
    3. Paolo Annibale & Stefano Vanni & Marco Scarselli & Ursula Rothlisberger & Aleksandra Radenovic, 2011. "Quantitative Photo Activated Localization Microscopy: Unraveling the Effects of Photoblinking," PLOS ONE, Public Library of Science, vol. 6(7), pages 1-8, July.
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    1. Steven J Tobin & Eliedonna E Cacao & Daniel Wing Wo Hong & Lars Terenius & Vladana Vukojevic & Tijana Jovanovic-Talisman, 2014. "Nanoscale Effects of Ethanol and Naltrexone on Protein Organization in the Plasma Membrane Studied by Photoactivated Localization Microscopy (PALM)," PLOS ONE, Public Library of Science, vol. 9(2), pages 1-7, February.

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