Last year, 29 researchers used the resources of the Laboratory of Cellular and Molecular Biology Microscopy Core. While most of the researchers come from the Laboratory of Cellular and Molecular Biology, the Core has been used by scientists from the Laboratory of Immune Cell Biology of the National Cancer Institute, the Cancer Immunobiology Section of the National Cancer Institute, the Section on Retinal Cell Biology and Degeneration at the National Eye Institute, the Genetic Disease Research Branch of the National Human Genome Research institute, and the University of Maryland Department of Physics. Almost all of the Principal Investigators in the Laboratory of Cellular and Molecular Biology have projects that involve the Core facility. Dr. Lawrence Samelson uses Core resources for the project Biochemical Basis of T Cell Activation. Dr. Carole Parent's projects, Signaling Events Regulating Chemotaxis and Chemotactic Signals Regulating Human Neutrophil and Breast Metastatic Migration used Core instruments. Dr. Paul Randazzo has made extensive use of the Core for the projects Regulation of focal adhesions and Turnover of invadopodia. Dr. Ying Zhang uses Core instruments for the project Molecular Mechanisms of TGF-beta Signaling Pathway. Dr. Ramiro Iglesias-Bartolome uses Core instruments for two projects, Signaling pathways regulating stem cell fate decisions and G-protein-coupled receptor signaling in cancer development and treatment. In addition, the Core facility has been used by personnel working with Principal Investigators from other groups including work with Dr. Jonathan Ashwell on the role of ZAP-70 in T cell activation. This research usually involves the use of a Leica SP8 Laser Scanning Confocal Microscope or a Nikon Total Internal Reflection Fluorescence (TIRF) microscope, with some usage of our Spinning Disk Confocal Microscope. Most of the users view immunofluorescent staining on fixed samples with the Leica laser scanning confocal microscope. The spinning disk confocal is generally used for live cell imaging, although all of our systems are equipped with on-stage incubators so that live cell imaging can be done on any microscope. We routinely use our TIRF microscope for PhotoActivation Localization Microscopy (PALM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). These Single Molecule Localization techniques allow us to determine the position of single proteins with a localization error of around 20 nm for PALM and 3 nm for dSTORM. Dr. Jason Yi from LCMB and Dr. Keir Neuman from the Biochemistry and Biophysics Center at National Heart Lung and Blood Institute along with the LCMB Core personnel pioneered the use of nano-diamonds as fiducial markers in Single Molecule Localization Microscopy. We routinely perform two color PALM imaging and multiplexed 3-D dSTORM imaging of up to 25 different proteins. The Laboratory of Cellular and Molecular Biology Microscopy Core obtained a new state-of-the-art image processing station for large 4D data sets, including analysis of single molecule data. This year the LCMB Core personnel conducted an extensive study of the super-resolution microscopy systems available on the Bethesda NIH campus and presented the results to the LCMB researchers so that they could add these techniques to their current research projects.
| Guittard, Geoffrey; Dios-Esponera, Ana; Palmer, Douglas C et al. (2018) The Cish SH2 domain is essential for PLC-?1 regulation in TCR stimulated CD8+ T cells. Sci Rep 8:5336 |
| Dutta, Debjani; Barr, Valarie A; Akpan, Itoro et al. (2017) Recruitment of calcineurin to the TCR positively regulates T cell activation. Nat Immunol 18:196-204 |
| Barr, Valarie A; Yi, Jason; Samelson, Lawrence E (2017) Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule Localization Microscopy. Methods Mol Biol 1584:183-206 |
| Yi, Jason; Manna, Asit; Barr, Valarie A et al. (2017) Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM. J Vis Exp : |
| Sherman, Eilon; Barr, Valarie A; Merrill, Robert K et al. (2016) Hierarchical nanostructure and synergy of multimolecular signalling complexes. Nat Commun 7:12161 |
| Barr, Valarie A; Sherman, Eilon; Yi, Jason et al. (2016) Development of nanoscale structure in LAT-based signaling complexes. J Cell Sci 129:4548-4562 |
| Yi, Jason; Manna, Asit; Barr, Valarie A et al. (2016) madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy. Mol Biol Cell 27:3591-3600 |
| Balagopalan, Lakshmi; Kortum, Robert L; Coussens, Nathan P et al. (2015) The linker for activation of T cells (LAT) signaling hub: from signaling complexes to microclusters. J Biol Chem 290:26422-9 |
| Zvezdova, Ekaterina; Lee, Jan; El-Khoury, Dalal et al. (2014) In vivo functional mapping of the conserved protein domains within murine Themis1. Immunol Cell Biol 92:721-8 |
| Sherman, Eilon; Barr, Valarie; Samelson, Lawrence E (2013) Super-resolution characterization of TCR-dependent signaling clusters. Immunol Rev 251:21-35 |
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