Conventional light and fluorescence microscopy techniques have offered tremendous insight into cellular processes and structures. Their resolution is however intrinsically limited by diffraction. Superresolution techniques achieve an order of magnitude higher resolution. Among these, localization microscopy relies on the position determination of single emitters with nanometer accuracy, which allows the subsequent reconstruction of an image of the target structure. In this chapter, we provide general guidelines for localization microscopy with a focus on Saccharomyces cerevisiae. Its different cellular architecture complicates efforts to directly transfer protocols established in mammalian cells to yeast. We compare different methodologies to label structures of interest and provide protocols for the respective sample preparation, which are not limited to yeast. Using these guidelines, nanoscopic subcellular structures in yeast can be investigated by localization microscopy, which perfectly complements live-cell fluorescence and electron microscopy.
Keywords: Diffraction limit; Localization microscopy; Saccharomyces cerevisiae; Sample preparation; Superresolution imaging.
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