Background: Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings: Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions: Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.

Department of Radioelectronics Faculty of Electrical Engineering Czech Technical University Prague Technická 2 16627 Prague 6 Czech Republic

Department of Radioelectronics Faculty of Electrical Engineering Czech Technical University Prague Technická 2 16627 Prague 6 Czech Republic Laboratory of Nanoscale Biology École Polytechnique Fédérale de Lausanne CH 1015 Lausanne Switzerland

UCCS Center for the Biofrontiers Institute University of Colorado at Colorado Springs 1420 Austin Bluffs Parkway Colorado Springs Colorado 80918 USA

UCCS Center for the Biofrontiers Institute University of Colorado at Colorado Springs 1420 Austin Bluffs Parkway Colorado Springs Colorado 80918 USA Department of Physics and Energy Science University of Colorado at Colorado Springs 1420 Austin Bluffs Parkway Colorado Springs Colorado 80918 USA

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