Abstract
Over the past years several forms of superresolution fluorescence microscopy have been developed that offer the possibility to study cellular structures and protein distribution at a resolution well below the diffraction limit of conventional fluorescence microscopy (<200 nm). A particularly powerful superresolution technique is single-molecule localization microscopy (SMLM). SMLM enables the quantitative investigation of subcellular protein distribution at a spatial resolution up to tenfold higher than conventional imaging, even in live cells. Not surprisingly, SMLM has therefore been used in many applications in biology, including neuroscience. This chapter provides a step-by-step SMLM protocol to visualize the nanoscale organization of endogenous proteins in dissociated neurons but can be extended to image other adherent cultured cells. We outline a number of methods to visualize endogenous proteins in neurons for live-cell and fixed application, including immunolabeling, the use of intrabodies for live-cell SMLM, and endogenous tagging using CRISPR/Cas9.
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Acknowledgments
This work was supported by the Netherlands Organization for Scientific Research (ALW-VIDI 171.029 to H.D.M.) and the European Research Council (ERC-StG 716011 to H.D.M.).
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Willems, J., Westra, M., MacGillavry, H.D. (2022). Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_16
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DOI: https://doi.org/10.1007/978-1-0716-2051-9_16
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