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QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ

Nature Methods volume 7, pages 339–340 (2010)Cite this article

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Although conventional microscopes have a reso-lution limited by diffraction to about half the wavelength of light, several recent advances have led to microscopy methods that achieve roughly tenfold improvements in resolution. Among them, photoactivated light microscopy (PALM) and stochastic optical resolution microscopy (STORM) have become particularly popular, as they only require relatively simple and affordable modifications to a standard total internal reflection fluorescence (TIRF) microscope1,2,3 and have been extended to three-dimensional (3D) super-resolution and multicolor imaging4,5. PALM and STORM achieve super-resolution by sequentially imaging sparse subsets of photoswitchable molecules. Positions of individual molecules are computed from individual low-resolution images with subdiffraction accuracy. These positions are then corrected for drifts and subsequently assembled into one or more super-resolution images. Unfortunately, in most current implementations, this reconstruction may take from several hours to days for a single dataset, thus forbidding visual inspection of super-resolution images in real time. Additionally, PALM and STORM software used to date is generally not freely available, strongly limiting the adoption of this otherwise relatively simple microscopy method. Two recent publications independently demonstrated two-dimensional (2D) algorithms for real-time PALM and STORM reconstructions6,7. Here we present QuickPALM (Supplementary Software 1), a freely available and open-source algorithm as a plugin for the widely used ImageJ (http://rsb.info.nih.gov/ij/) software that combines real-time processing capability with additional important features including 3D reconstruction, drift correction and real-time acquisition control (http://code.google.com/p/quickpalm/).

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Figure 1: Three-dimensional dSTORM imaging of β-III-tubulin–labeled microtubules in neuronal cells.

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Acknowledgements

We thank J. Enninga for assistance with experiments, and T. Duong and R. Fesce for helpful insight in the preparation of the manuscript.

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Authors and Affiliations

  1. Unidade de Biofísica e Expressão Genética, Instituto de Medicina Molecular, Faculdade de Medicina Universidade de Lisboa, Lisboa, Portugal

    Ricardo Henriques & Musa M Mhlanga

  2. Institut Pasteur, Groupe Imagerie et Modélisation, Institut Pasteur, Groupe Imagerie et Modélisation; Centre National de la Recherche Scientifique,

    Ricardo Henriques, Mickael Lelek & Christophe Zimmer

  3. Centre National de la Recherche Scientifique, Unité de Recherche Associée 2582, Paris, France

    Ricardo Henriques, Mickael Lelek & Christophe Zimmer

  4. Istituto Scientifico San Raffaele, Università Vita-Salute, Milano, Italia

    Eugenio F Fornasiero & Flavia Valtorta

  5. Unità di Neuroscienze Molecolari, Istituto Italiano di Tecnologia, Milano, Italia

    Eugenio F Fornasiero & Flavia Valtorta

  6. Gene Expression and Biophysics Group, Synthetic Biology Emerging Research Area, Council for Scientific and Industrial Research, Pretoria, South Africa

    Musa M Mhlanga

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  1. Ricardo Henriques
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Correspondence to Ricardo Henriques.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2, Supplementary Methods, Supplementary Note 1, Supplementary Discussion (PDF 903 kb)

Supplementary Software 1

QuickPALM software (ZIP 5714 kb)

Supplementary Software 2

Laser control software (ZIP 5596 kb)

Supplementary Movie 1

A 3D presentation of microtubules shown in Figure 1e,f. The viewing angle is rotated here to show different perspectives. Scale bar, 1,000 nm. (MOV 1376 kb)

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Henriques, R., Lelek, M., Fornasiero, E. et al. QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ. Nat Methods 7, 339–340 (2010). https://doi.org/10.1038/nmeth0510-339

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