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Nat Methods. 2015 Aug;12(8):717-24. doi: 10.1038/nmeth.3442. Epub 2015 Jun 15.

Quantitative evaluation of software packages for single-molecule localization microscopy.

Author information

Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Center for Genomic Regulation, Barcelona, Spain.
1] Howard Hughes Medical Institute, University of California (UCSF), San Francisco, California, USA. [2] Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, California, USA.
Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea.
Laboratory of Experimental Biophysics, EPFL, Lausanne, Switzerland.


The quality of super-resolution images obtained by single-molecule localization microscopy (SMLM) depends largely on the software used to detect and accurately localize point sources. In this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. Our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. These metrics reflect the various tradeoffs of SMLM software packages and help users to choose the software that fits their needs.

[Indexed for MEDLINE]

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