Abstract
Single-molecule localization microscopy (SMLM) comprises methods that produce super-resolution images from molecular locations of single molecules. These techniques mathematically determine the center of a diffraction-limited spot produced by a fluorescent molecule, which represents the most likely location of the molecule. Only a small cohort of well-separated molecules is visualized in a single image, and then many images are obtained from a single sample. The localizations from all the images are combined to produce a super-resolution picture of the sample. Here we describe the application of two methods, photoactivation localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM), to the study of signaling microclusters in T cells.
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Acknowledgment
We thank Eilon Sherman for generating the algorithms used for our PALM analysis and continued advice on imaging methods. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute (NCI), and Center for Cancer Research.
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Barr, V.A., Yi, J., Samelson, L.E. (2017). Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_13
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DOI: https://doi.org/10.1007/978-1-4939-6881-7_13
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