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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. © 2014 Springer Science+Business Media, LLC.

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167 - 188