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Artifacts in single-molecule localization microscopy

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Abstract

Single-molecule localization microscopy provides subdiffraction resolution images with virtually molecular resolution. Through the availability of commercial instruments and open-source reconstruction software, achieving super resolution is now public domain. However, despite its conceptual simplicity, localization microscopy remains prone to user errors. Using direct stochastic optical reconstruction microscopy, we investigate the impact of irradiation intensity, label density and photoswitching behavior on the distribution of membrane proteins in reconstructed super-resolution images. We demonstrate that high emitter densities in combination with inappropriate photoswitching rates give rise to the appearance of artificial membrane clusters. Especially, two-dimensional imaging of intrinsically three-dimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the single-molecule movies recorded to reconstruct the images have to be carefully investigated especially when investigating membrane organization and cluster analysis.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, Grant No. SA 829/13-9). We thank Sebastian Malkusch for providing us the cluster analysis software Lama.

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    Authors and Affiliations

    1. Department of Biotechnology and Biophysics, University Würzburg, Am Hubland, 97074, Würzburg, Germany

      Anne Burgert, Sebastian Letschert, Sören Doose & Markus Sauer

    Authors
    1. Anne Burgert
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    2. Sebastian Letschert
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    3. Sören Doose
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    4. Markus Sauer
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    Corresponding authors

    Correspondence to Sören Doose or Markus Sauer.

    Additional information

    Anne Burgert and Sebastian Letschert have contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    dSTORM movie_1

    Microtubules labeled U2OS cells shown in Fig. 1 excited at 641 nm with ~7 kW cm−2 at a frame rate of 50 Hz (AVI 889 kb)

    dSTORM movie_2

    Microtubules labeled U2OS cells shown in Fig. 1 excited at 641 nm with ~0.3 kW cm−2 at a frame rate of 50 Hz. (AVI 1574 kb)

    dSTORM movie_3

    Microtubules labeled U2OS cells shown in Fig. 1 excited at 641 nm with ~0.1 kW cm−2 at a frame rate of 50 Hz. (AVI 1585 kb)

    dSTORM movie_4

    Alexa Fluor 647-WGA-labeled glycans in the basal plasma membrane of U2OS cells shown in Fig. 1 excited at 641 nm with ~7 kW cm−2 at a frame rate of 50 Hz. (AVI 1879 kb)

    dSTORM movie_5

    Alexa Fluor 647-WGA-labeled glycans in the basal plasma membrane of U2OS cells shown in Fig. 1 excited at 641 nm with ~0.3 kW cm−2 at a frame rate of 50 Hz. (AVI 2394 kb)

    dSTORM movie_6

    Alexa Fluor 647-WGA-labeled glycans in the basal plasma membrane of U2OS cells shown in Fig. 1 excited at 641 nm with ~0.1 kW cm−2 at a frame rate of 50 Hz. (AVI 2456 kb)

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    Burgert, A., Letschert, S., Doose, S. et al. Artifacts in single-molecule localization microscopy. Histochem Cell Biol 144, 123–131 (2015). https://doi.org/10.1007/s00418-015-1340-4

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    • DOI: https://doi.org/10.1007/s00418-015-1340-4

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