Immunocompromise of the T cell response may facilitate the aging process; thus, understanding the spatio-temporal dynamics of signaling in T cells at the molecular level is crucial. We hypothesize that cholesterol-rich membrane domains (or ?rafts?) facilitate functionally relevant compartmentalization for effective T cell signaling, and these critical molecular interactions between the T cell receptor and other signaling proteins and specific lipids are disrupted as aging proceeds. Cholesterol-rich rafts are hypothesized to be transient and microscopic and, until now, have not yet been directly observed in vivo without significant perturbation. This interdisciplinary NIH R21 proposal will overcome this problem by combining cutting-edge imaging tools with experimental approaches that will allow us to understand quantitatively how molecules interact in biomembranes. To test our hypothesis, we propose the following two Specific Aims: Specific Aim 1: Investigating the signaling dynamics of lipid- and protein-induced functional domains in young and aging T cell models with high spatial and temporal resolution using one- and two-photon micro-spectroscopy; and Specific Aim 2: Investigating the chemical nature of membrane domains that participate in immunoreceptor signaling using imaging mass spectrometry. For our initial studies on the latter Specific Aim, we will use the related model system of IgE receptor signaling in RBL mast cells, which serves as a robust platform for the development of imaging mass spectrometry on intact cells that we will subsequently use on the T cell models. The projected results will lead to new insights into how T cell receptor signaling dynamics and related plasma membrane nanostructure change as T cells age.[unreadable] [unreadable] [unreadable]