(Provided by the applicant) Abstract: The inability of current super-resolution methods to generate multi color in situ images and dynamic information limits their impact. The challenge at hand is to advance this purely descriptive technology to study the mechanisms of highly dynamic and rare molecular processes. We will approach the task of visualizing two different colors in live specimens through the combination of different fluorophores and new acquisition strategies. Information on the interplay between molecule distributions and movement will be derived from simultaneous analyses of molecule dynamics by fluorescence cross correlation spectroscopy. With this novel technology, we will study the architecture of the plasma membrane and its effects on membrane associated signaling. This research will provide visual and mechanistic insights necessary to develop a unified plasma membrane theory. Specifically, we will explore the segregation of all membrane proteins into domains and the basis of their distinct confinement. In addition, the proposed research wil disect the spatio-temporal mechanisms of different signaling pathways (TCR, EGF and cytokine signaling). We expect to discover novel control principles in signaling that are rooted in the architecture of the plasma membrane. Thus, our studies have the potential to revolutionize our understanding of the molecular underpinnings regulating membrane- associated signaling. This is crucially important in view of the large number of diseases associated with membrane signaling defects. Our studies will verify the potential for the modulation of membrane associated signaling through changes in the plasma membrane organization. Public Health Relevance: Studying the plasma membrane structure and signaling mechanisms rooted in it lays the foundation for insights into a large number of diseases that are caused by defects in membrane signaling. The proposed research aims to establish technology that can determine the spatial and dynamic underpinnings of membraneassociated signaling based on super-resolution and cross-correlation optical microscopy. It will provide new avenues for modulating cellular signaling through changes in its environment, a more 'gentle'adjustment without causing the dramatic side effects of current approaches.