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Huygens Localizer


Several additional tools are included in


Huygens Localizer. For example, results can be analyzed in more detail using the Huygens Object Analyzer module, which allows iso- surface-based quantitative measurements in the super-resolved images. Te latest addition is the Huygens Batch Processor, which allows high-throughput analysis of SMLM data.


Conclusion In this paper we presented Huygens Local-


izer for analyzing and visualizing SMLM data. Huygens Localizer provides researchers with a well-supported, easy-to-use, reliable, accurate, and very fast solution for the entire SMLM workflow. A demo version of Huygens Local- izer can be downloaded from https://www.svi. nl/Download.


Acknowledgments We thank Mikko Liljeström (Biomedicum


Figure 8: 3D SMLM analysis of the mitochondrial import receptor subunit TOM20 in human bone osteosarcoma epithelial (U2OS) cells using DNA-PAINT. (A) 3D SFP rendering of the 3D result. The size of the rendered volume is 10 μm × 10 μm × 1 μm. The inset shows a rendering of the globular structure indicated with the white arrow at a higher resolution, cut by a Z-plane through the middle of the structure. The step size of the shown grid is 200 nm. (B) Result of the 2D SMLM analysis of the data. (C) A single slice through the middle of the result of the 3D SMLM analysis showing effective depth discrimination of structures above or below the slice. Scalebar: 2 μm.


techniques where the image is directly formed by imaging on a camera, or by scanning the sample. Instead, a time-series of sparse 2D images is acquired where the individual fluorophores can be distinguished. Tese data are processed to obtain a table of fluorophore locations together with a super-resolved image of the object. In the case of 3D SMLM, analysis is even more com- plicated due to the need to calibrate the Z position at a nanome- ter scale. Compared to direct imaging techniques, this imposes stricter requirements on the post-processing soſtware, which must provide accurate localization results and visualizations, preferably at high speed. Huygens Localizer fulfills these requirements by offering


a user-friendly, wizard-driven interface to help with the com- plete SMLM data analysis pipeline, from background detection to rendering the super-resolved image. Using highly optimized multi-core CPU and GPU code, SMLM data can be analyzed and visualized quickly and accurately. Huygens Localizer includes the PSF Distiller, a time-tested module from the Huy- gens deconvolution package [7], which has been adapted to enable robust calibration of 3D SMLM data.


2020 March • www.microscopy-today.com


Imaging Unit, University of Helsinki) for pro- viding the data used in Figures 5 and 6. We thank Marko Lampe (Advanced Light Micros- copy Facility, European Molecular Biology Lab- oratory, Heidelberg, Germany) for recording the data used in Figures 7 and 8 and Christoph Spahn and Mike Heilemann (Goethe Univer- sity Frankfurt, Frankfurt am Main, Germany) for preparing the TOM20 DNA-PAINT sample. We thank Gert van Cappellen and Johan Slot- man (Erasmus Medical Center, Rotterdam, the Netherlands) for helpful discussions and their great patience in evaluating early versions of the Huygens Localizer. We thank the LCI Resource Laboratory, University of Calgary, for their support and imaging advice.


References [1] E Betzig et al., Science 313 (2006) 1642–45. [2] ST Hess et al., Biophys J 91 (2006) 4258–72. [3] MJ Rust et al., Nat Methods 3 (2006) 793–95. [4] M Heilemann et al., Angew Chem Int Ed 47 (2008) 6172–76. [5] J Fölling et al., Nat Methods 5 (2008) 943–45. [6] A Sharonov and RM Hochstrasser, PNAS 103 (2006) 18911–16. [7] Huygens soſtware is described at this website: Scientific Volume Imaging, “Deconvolution - Visualization - Anal- ysis.” https://svi.nl (accessed January 11, 2020).


[8] D Sage et al., Nat Methods 12 (2015) 717–24. [9] D Sage et al., Nat Methods 16 (2019) 387–95.


[10] B Huang et al., Science 319 (2008) 810–13. [11] SRP Pavani et al., PNAS 106 (2009) 2995–99. [12] Huygens Localizer white paper, https://svi.nl/WhitePapers, HuygensLocalizerWhitePaper.pdf 2020).


(accessed January


[13] E Wegel et al., Sci Rep 6 (2016) 27290. [14] J Schnitzbauer et al., Nat Protoc 6 (2017) 1198–228.


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