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 Physics and Energy Science University of Colorado at Colorado Springs 1420 Austin Bluffs Parkway Colorado Springs Colorado 80918 USA

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

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Betzig E, Patterson GH, Sougrat R, et al. . Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006;313:1642–5. PubMed

Hess ST, Girirajan TPK, Mason MD. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006;91:4258–72. PubMed PMC

Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006;3:793–5. PubMed PMC

Heilemann M, van de Linde S, Schüttpelz M, et al. . Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew Chemie Int Ed. 2008;47:6172–6. PubMed

Dertinger T, Colyer R, Iyer G et al. . Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc Natl Acad Sci U S A. 2009;106:22287–92. PubMed PMC

Geissbuehler S, Bocchio NL, Dellagiacoma C et al. . Mapping molecular statistics with balanced super-resolution optical fluctuation imaging (bSOFI). Opt Nanoscopy. 2012;1:4.

Hell SW, Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 1994;19:780. PubMed

Heintzmann R, Cremer C. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc SPIE. 1998;3568:185–96.

Gustafsson MGL. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000;198:82–87. PubMed

Neil MAA, Juškaitis R, Wilson T. Method of obtaining optical sectioning by using structured light in a conventional microscope. Opt Lett. 1997;22:1905–7. PubMed

Gustafsson MGL, Shao L, Carlton PM, et al. . Three-dimensional resolution doubling in widefield fluorescence microscopy by structured illumination. Biophys J. 2008;94:4957–70. PubMed PMC

Kner P, Chhun BB, Griffis ER et al. . Super-resolution video microscopy of live cells by structured illumination. Nat Methods. 2009;6:339–42. PubMed PMC

Hirvonen LM, Wicker K, Mandula O et al. . Structured illumination microscopy of a living cell. Eur Biophys J. 2009;38:807–12. PubMed

Shao L, Kner P, Rego EH, et al. . Super-resolution 3D microscopy of live whole cells using structured illumination. Nat Methods. 2011;8:1044–6. PubMed

Schermelleh L, Carlton PM, Haase S et al. . Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science. 2008;320:1332–6. PubMed PMC

Fiolka R, Shao L, Rego EH et al. . Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc Natl Acad Sci U S A. 2012;109:5311–5. PubMed PMC

York AG, Parekh SH, Nogare DD et al. . Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat Methods. 2012;9:749–54. PubMed PMC

Ingaramo M, York AG, Wawrzusin P et al. . Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue. Proc Natl Acad Sci U S A. 2014;111:5254–9. PubMed PMC

York AG, Chandris P, Nogare DD et al. . Instant super-resolution imaging in live cells and embryos via analog image processing. Nat Methods. 2013;10:1122–6. PubMed PMC

Schropp M, Uhl R. Two-dimensional structured illumination microscopy. J Microsc. 2014;256:23–36. PubMed

Schropp M, Seebacher C, Uhl R. XL-SIM: Extending superresolution into deeper layers. Photonics. 2017;4:33.

Planchon TA, Gao L, Milkie DE, et al. . Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat Methods. 2011;8:417–23. PubMed PMC

Keller PJ, Schmidt AD, Santella A, et al. . Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Meth. 2010;7:637–42. PubMed PMC

Gao L, Shao L, Higgins CCD et al. . Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell. 2012;151:1370–85. PubMed PMC

Chen B-C, Legant WR, Wang K et al. . Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science. 2014;346:1257998. PubMed PMC

Li D, Shao L, Chen B-C et al. . Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science. 2015;349:aab3500. PubMed PMC

Dong S, Nanda P, Shiradkar R, et al. . High-resolution fluorescence imaging via pattern-illuminated Fourier ptychography. Opt Express. 2014;22:20856–70. PubMed

Orieux F, Sepulveda E, Loriette V et al. . Bayesian estimation for optimized structured illumination microscopy. IEEE Trans Image Process. 2012;21:601–14. PubMed

Lukeš T, Hagen GM, Křížek P et al. . Comparison of image reconstruction methods for structured illumination microscopy. Proc SPIE. 2014;9129:91293J.

Lukeš T, Křížek P, Švindrych Z, et al. . Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation. Opt Express. 2014;22:29805–17. PubMed

Mudry E, Belkebir K, Girard J, et al. . Structured illumination microscopy using unknown speckle patterns. Nat Photonics. 2012;6:312–5.

Huang X, Fan J, Li L, et al. . Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy. Nat Biotechnol. 2018, 36, 451–459. PubMed

Perez V, Chang BJ, Stelzer EHK. Optimal 2D-SIM reconstruction by two filtering steps with Richardson-Lucy deconvolution. Sci Rep. 2016;6:37149. PubMed PMC

Chu K, McMillan PJ, Smith ZJ, et al. . Image reconstruction for structured-illumination microscopy with low signal level. Opt Express. 2014;22:8687. PubMed

Chakrova N, Rieger B, Stallinga S. Deconvolution methods for structured illumination microscopy. J Opt Soc Am A. 2016;33:B12. PubMed

Křížek P, Lukeš T, Ovesný M et al. . SIMToolbox: a MATLAB toolbox for structured illumination fluorescence microscopy. Bioinformatics. 2015;32:318–20. PubMed

Křížek P, Raška I, Hagen GM. Flexible structured illumination microscope with a programmable illumination array. Opt Express. 2012;20:24585. PubMed

Hagen GM, Caarls W, Thomas M et al. . Biological applications of an LCoS-based programmable array microscope. Proc SPIE. 2007;64410S:1–12.

Werley CA, Chien M-P, Cohen AE. An ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation. Biomed Opt Express. 2017;8:5794. PubMed PMC

Dan D, Lei M, Yao B, et al. . DMD-based LED-illumination super-resolution and optical sectioning microscopy. Sci Rep. 2013;3:1116. PubMed PMC

Křížek P, Hagen GM. Spatial light modulators in fluorescence microscopy. In Microscopy: Science, Technology, Applications and Education, Méndez-Vilas A.ed., 4th ed (Formatex, 2010), Vol. 2, pp. 1366–77.

Hagen GM, Caarls W, Lidke KA et al. . Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope. Microsc Res Tech. 2009;72:431–40. PubMed PMC

Song L, Lu-Walther H-W, Förster R et al. . Fast structured illumination microscopy using rolling shutter cameras. Meas Sci Technol. 2016;27:055401.

Cvačková Z, Mašata M, Stanĕk D, et al. . Chromatin position in human HepG2 cells: although being non-random, significantly changed in daughter cells. J Struct Biol. 2009;165:107–17. PubMed PMC

Young LJ, Ströhl F, Kaminski CF. A guide to structured illumination TIRF microscopy at high speed with multiple colors. J Vis Exp. 2016;e53988. PubMed PMC

Förster R, Lu-Walther H-W, Jost A, et al. . Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator. Opt Express. 2014;22:20663–77. PubMed

Lu-Walther H-W, Kielhorn M, Förster R et al. . fastSIM: a practical implementation of fast structured illumination microscopy. Methods Appl Fluoresc. 2015;3:014001. PubMed

Schlichenmeyer TC, Wang M, Elfer KN et al. . Video-rate structured illumination microscopy for high-throughput imaging of large tissue areas. Biomed Opt Express. 2014;5:366–77. PubMed PMC

Kantelhardt SR, Caarls W, de Vries AHB et al. . Specific visualization of glioma cells in living low-grade tumor tissue. PLoS One. 2010;5:1–11. PubMed PMC

SIM Toolbox http://mmtg.fel.cvut.cz/SIMToolbox. Accessed November 6, 2018.

Pospíšil J, Fliegel K, Klíma M. Assessing resolution in live cell structured illumination microscopy. In Proceedings of SPIE - The International Society for Optical Engineering, Páta P, Fliegel Keds. (SPIE, 2017), Vol. 10603, p. 39.

Agarwal AK, Srinivasan N, Godbole R et al. . Role of tumor cell surface lysosome-associated membrane protein-1 (LAMP1) and its associated carbohydrates in lung metastasis. J Cancer Res Clin Oncol. 2015;141:1563–74. PubMed

http://mmtg.fel.cvut.cz/mapsimlive_suppl/. Supplementary Video, accessed November 6, 2018.

Pospíšil J, Lukeš T, Bendesky J, et al. . Supporting data for “Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction.”. GigaScience Database. 2018, 10.5524/100514. PubMed DOI PMC

Geissbuehler M, Lasser T. How to display data by color schemes compatible with red-green color perception deficiencies. Opt Express. 2013;21:9862–74. PubMed

Huang F, Hartwich TMP, Rivera-Molina FE et al. . Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat Methods. 2013;10:1–9. PubMed PMC

Goodman JW. Frequency Analysis of Optical Imaging Systems. In Introduction to Fourier Optics, 2nd ed (McGraw-Hill Int, 1968), pp. 126–71.

Open-source microscope add-on for structured illumination microscopy

Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction