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ViSP: representing single-particle localizations in three dimensions

Nature Methods volume 10, pages 689–690 (2013)Cite this article

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To the Editor:

Recent developments in three-dimensional (3D) methods of localizing stochastically activated fluorescent probes have advanced our understanding of the organization of biological systems at the nanoscale level1. Techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) make it possible to probe the morphology of subcellular structures and detail their dynamic behavior in three dimensions (with spatial resolutions as small as 10 nm (ref. 2)). Although powerful programs for localizing 3D single-particle detections are publicly available (such as QuickPALM3, rapidSTORM4 and the μManager localization microscopy plug-in), user-friendly tools for representing generated localization data in a biologically relevant manner remain in great demand in the super-resolution microscopy community. In this regard, among the greatest challenges include the efficient handling of millions of individual localizations; the description of localization precisions in three dimensions; and the incorporation of intuitive depth cues, multichannel compatibilities and 3D quantitative features. With no obvious solutions in either the commercial or public software domains, we developed ViSP: an interactive, freely available, cross-platform 3D localization representation tool (Supplementary Software).

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Figure 1: Visualization of 3D single-particle localizations with ViSP.

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Acknowledgements

We thank B. Hajj (Institut Curie), J. Chen (Janelia Farm Research Campus), I. Izeddin (Ecole Normale Supérieure), J. Sillibourne (Commissariat à l'Energie Atomique et aux Energies Alternatives) and M. Bornens (Institut Curie) for providing super-resolution data sets used in the figures of this correspondence. We are grateful to K. Busch (Universität Osnabrück) for sharing the plasmid TOM-20-GFP. We also thank N. Clack, F. Mueller and X. Darzacq for discussions during the preparation of this work. This work was supported by funding from the Transcription Imaging Consortium at the Janelia Farm Research Campus, the Institut Curie International PhD Program and by the Tridimic grant from the program Recherches Partenariales et Innovation Biomédicale from Agence Nationale pour la Recherche.

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Authors and Affiliations

  1. Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Paris, France

    Mohamed El Beheiry & Maxime Dahan

  2. Transcription Imaging Consortium, Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA

    Mohamed El Beheiry & Maxime Dahan

Authors
  1. Mohamed El Beheiry
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  2. Maxime Dahan
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Correspondence to Mohamed El Beheiry or Maxime Dahan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Software

ViSP executables for Windows and Mac OS X (with User Manual and License) (ZIP 15640 kb)

Comparison of isotropic and anisotropic representation of 3D STORM localizations

Rotation of a 3D STORM acquired mammalian mitochondria segment (U2OS cell transfected with TOM20-GFP and nanobody label with Alexa 647) to illustrate differences in localization precision between the lateral and axial dimensions using ViSP. The left panel is a reconstruction with isotropic Gaussians, while the right panel is that of a reconstruction with anisotropic Gaussians, revealing a poorer localization precision in the axial (z) dimension. Size of region is 1.4 × 3.2 × 1.8 μm. (MOV 3790 kb)

Cluster segmentation of 3D STORM mammalian mitochondria network

Left panel shows a 200,000 localization reconstruction of a 3D STORM acquired mammalian mitochondria network (U2OS cell transfected with TOM20-GFP and nanobody labeling with Alexa 647). Right panel is a surface rendering and cluster segmentation of the mitochondria after a local density threshold using ViSP. (MOV 27813 kb)

Comparison of localization- and surface-based representation of 3D PALM/STORM multi-channel data

Left panel is a localization-based representation of a multi-channel centrosome complex (mEos2-Centrin1 in red and Alexa 647-labelled Cep 164 in blue) in a combined 3D PALM/STORM acquisition. Right panel is the representation of the same channels using ViSP's surface rendering feature. (MOV 13957 kb)

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Beheiry, M., Dahan, M. ViSP: representing single-particle localizations in three dimensions. Nat Methods 10, 689–690 (2013). https://doi.org/10.1038/nmeth.2566

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