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Online image analysis software for photoactivation localization microscopy

Nature Methods volume 6, pages 689–690 (2009)Cite this article

To the Editor: In photoactivation localization microscopy (PALM), super-resolution imaging is achieved by precise localization of many photoactivatable fluorophores. Image analysis by a fit procedure has so far been much slower than data acquisition. Here we report a software program that speeds up fluorophore localization by a factor of ∼200 while maintaining a comparable image quality. This online analysis renders PALM an even more powerful imaging method.

In recent years, ingenious super-resolution optical microscopy (nanoscopy) techniques have been devised that circumvent the diffraction barrier, which normally restricts the image resolution to ∼200 nm (ref. 1). PALM is a wide-field technique based on the detection of the fluorescence emission from individual fluorophores that can be photoactivated by irradiation with visible light2,3,4. A few fluorophores are switched on by a light pulse, an image is taken, and the molecules are subsequently switched off reversibly (deactivated) or irreversibly (photobleached). This procedure is repeated many times until a few thousand frames are collected. A final high-resolution image is reconstructed from the precise locations of the fluorophores in all frames, which can be determined to within tens of nanometers by a two-step algorithm. First, peaks corresponding to individual fluorophores are identified within each frame (searching step), and then their centers of mass are determined (localization step).

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Figure 1: Comparison of PALM image analysis using Gaussian fit localization and the fluoroBancroft algorithm.

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Acknowledgements

We thank S.T. Hess (University of Maine, Orono) for making his fluorescence PALM image analysis program available to us. This work was supported by the Deutsche Forschungsgemeinschaft and the State of Baden-Württemberg through the Center for Functional Nanostructures, by Deutsche Forschungsgemeinschaft grants NI 291/9 and SFB 497, and by the Fonds der Chemischen Industrie.

Author information

Authors and Affiliations

  1. Institute of Biophysics, University of Ulm, Ulm, Germany

    Per Niklas Hedde, Jochen Fuchs & Gerd Ulrich Nienhaus

  2. Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany

    Franz Oswald

  3. National Oceanography Center, University of Southampton, Southampton, UK

    Jörg Wiedenmann

  4. Institute of Applied Physics and Center for Functional Nanostructures, Universität Karlsruhe (TH) and Karlsruhe Institute of Technology, Karlsruhe, Germany

    Gerd Ulrich Nienhaus

  5. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Gerd Ulrich Nienhaus

Authors
  1. Per Niklas Hedde
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  2. Jochen Fuchs
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  3. Franz Oswald
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  4. Jörg Wiedenmann
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  5. Gerd Ulrich Nienhaus
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Corresponding author

Correspondence to Gerd Ulrich Nienhaus.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Note (PDF 1046 kb)

Supplementary Video 1

Live PALM display in real time. Individual frames as recorded by the CCD camera (left), sum of all recorded frames (middle), and positions of all localized molecules (right). An area of 40 × 40 pixels is shown, covering 5.2 × 5.2 μm of the sample. The display update interval can be varied and was here set to 1 s. (AVI 2984 kb)

Supplementary Software

Livepalm software. (ZIP 534 kb)

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Hedde, P., Fuchs, J., Oswald, F. et al. Online image analysis software for photoactivation localization microscopy. Nat Methods 6, 689–690 (2009). https://doi.org/10.1038/nmeth1009-689

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