The adapter molecule linker for activation of T cells (LAT) plays a crucial role in forming signaling complexes induced by stimulation of the T cell receptor (TCR). of LAT also increases at the same time. Both changes require TCR activation and an intact actin cytoskeleton. These results demonstrate that this nanoscale business of LAT-based signaling complexes is usually dynamic and indicates that different kinds of LAT-based complexes appear at different times during T cell activation. (Su et al., 2016). LAT-based oligomers appear to be important for activation of several downstream signaling pathways (Kortum et al., 2013). Grb2 can bind to any one of three tyrosine residues on LAT while simultaneously binding Sos1, and Sos1 can bind two Grb2 molecules, potentially forming CD271 a meshwork of cross-linked LAT molecules (Houtman et al., 2006; Kortum et al., 2013). Depletion of Grb2, loss of Sos1 or mutation of LAT to prevent multipoint Grb2 binding all cause decreased ERK activation, PLC-1 phosphorylation and diminished Ca2+ flux (Balagopalan et al., 2015). SLP-76 oligomers are also important for T cell activation. SLP-76 can be crosslinked by multipoint binding to the adapter protein ADAP (also known as FYB) at three phosphorylation sites (Boerth et al., 2000; da Silva et al., 1997). Removing two of these sites prevents crosslinking and leads to decreased Ca2+ flux. Thus, it appears that some level of oligomerization of LAT and SLP-76 is required to produce proper T cell activation (Coussens et al., 2013). Imaging studies have shown that TCR engagement leads to dramatic changes in T cells, including the rapid formation of discrete puncta termed microclusters (Balagopalan et al., 2011; Yokosuka and Saito, 2010). These microclusters have been studied extensively in T cells activated by peptideCMHC (pMHC) on an APC (Freiberg et al., 2002; Johnson et al., 2000; Krummel et al., 2000; Lee et al., 2002), through use of activating molecules incorporated into lipid bilayers (Campi et al., 2005; Grakoui et al., 1999; Ilani et al., 2009; Kaizuka et al., 2007; Yokosuka et al., 2005) and activating antibodies on glass surfaces (Barda-Saad et al., 2005; Bunnell Fexaramine et al., 2002, 2001). Microclusters initially contain most of the molecules required for TCR signaling, including both LAT and SLP-76 and they appear to be the sites where signal transduction begins (Bunnell et al., 2002; Varma et al., 2006; Yokosuka et al., 2005). Live-cell studies have shown that microclusters are dynamic structures, as constituents of the signaling complexes constantly dissociate and re-associate (Bunnell et al., 2002). Furthermore, the composition of signaling complexes changes as the cells spread; some Fexaramine proteins such as Gads and Cbl are only seen transiently in microclusters and are not present in microclusters visualized at later occasions (Balagopalan et al., Fexaramine 2007; Bunnell et al., 2002). To understand the dynamic business and potential heterogeneity of the signaling complexes induced by TCR engagement, we need to determine their molecular structures at various occasions after activation. Many researchers have turned to super-resolution microscopy techniques to observe molecular details beyond the diffraction limit of visible light (Nienhaus and Nienhaus, 2016; Sydor et al., 2015). Single-molecule localization microscopy (SMLM) has been used to visualize molecules found in microclusters at high resolution (Hsu and Baumgart, 2011; Lillemeier et al., 2010; Purbhoo et al., 2010; Rossy et al., 2013; Sherman et al., 2011). In SMLM, the center of a diffraction-limited spot produced by a single fluorescently labeled molecule is determined mathematically and defined as the probable location of the molecule (Allen et al., 2013; Knight, 2017). A small cohort of activated molecules is usually imaged and then they are photoswitched or photobleached. Another cohort of molecules can then be activated and the entire process is usually repeated many times to visualize thousands of single molecules. The position of each individual molecule is usually calculated from the corresponding diffraction-limited spot in the image series. These calculated positions, often called molecular peaks or localizations, are combined to produce an image showing the location of every visualized molecule. Two common methods are photo-activation localization microscopy (PALM) (Betzig et al., 2006; Sengupta et al., 2014) and direct.