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Application of Three-Dimensional Structured Illumination Microscopy in Cell Biology: Pitfalls and Practical Considerations

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Super-Resolution Microscopy Techniques in the Neurosciences

Part of the book series: Neuromethods ((NM,volume 86))

Abstract

Super-resolution fluorescence microscopy techniques have paved the way to address cell biological questions with unprecedented spatial resolution. Of these, three-dimensional structured illumination microscopy (3D-SIM) reaches a nearly eightfold increased volumetric resolution compared to conventional diffraction-limited methods and allows multicolor optical sectioning of standard fluorescently labeled fixed or live samples. Owing to its broad application spectrum, 3D-SIM is likely to become a key method in cell biological far-field imaging, complementing more specialized higher-resolving techniques, such as single molecule localization and cryo-electron microscopy. To fully explore the potential of 3D-SIM, however, considerably greater care needs to be taken with regard to the preparation of the sample, calibration of the instrument, post-processing of the data, and extraction of valid quantitative measurements. In this chapter we discuss technical problems typically encountered and provide guidelines for troubleshooting.

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Acknowledgments

The authors wish to thank Heinrich Leonhardt and the BioImaging Network Munich for generous support, Andreas Maiser for technical help, Fabio Spada and Markus Rehberg for providing samples, and Ian Dobbie, Justin Demmerle, and Yolanda Markaki for valuable comments on the manuscript. This work was supported by grants from the Center of Integrated Protein Science Munich and the DFG (TR5 and SCHE1596/2-1). The authors declare no conflict of interests.

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Authors and Affiliations

  1. Department of Biology, Ludwig Maximilians University Munich, Martinsried, Germany

    Daniel Smeets, Jürgen Neumann & Lothar Schermelleh

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Daniel Smeets & Lothar Schermelleh

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  1. Daniel Smeets
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  2. Jürgen Neumann
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  1. Department of Neuro- and Sensory Physiology,University of Göttingen Medical Center, Göttingen, Germany

    Eugenio F. Fornasiero

  2. Department of Neuro- and Sensory Physiology,University of Göttingen Medical Center, Göttingen, Germany

    Silvio O. Rizzoli

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Smeets, D., Neumann, J., Schermelleh, L. (2014). Application of Three-Dimensional Structured Illumination Microscopy in Cell Biology: Pitfalls and Practical Considerations. In: Fornasiero, E., Rizzoli, S. (eds) Super-Resolution Microscopy Techniques in the Neurosciences. Neuromethods, vol 86. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-983-3_8

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  • DOI: https://doi.org/10.1007/978-1-62703-983-3_8

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