Summary: Spectacular recent advances in single cell genomics have provided high-resolution information about cell identity, however relating molecular information to the spatial and temporal context remains a major challenge. This is particularly relevant for the immune system, since immune responses occur in highly organized tissue environments, and involve tightly-controlled changes in cell state over time. Here we propose to develop new approaches to increase the spatial and temporal resolution of single cell analyses, and to use these methods to generate and then disseminate a 4-dimensional (time and 3D spatial coordinates) map of T cell development and the associated microenvironment in the thymus. In Aim 1, we will use simultaneous measurement of mRNA and surface proteins on single cells, together with computation and experimental approaches to develop a temporal map of cell state transitions during T cell development in the thymus. In Aim 2, we will use coherent Raman and multiphoton microscopy of living thymic tissue slices, together with laser microdissection, to isolate functionally relevant regions of tissues. We will then perform single cell analyses of individual cells within these defined regions and use computational analyses to define cell types and resolve cellular cross-talk. In Aim 3, we will increase the value of this resource to the scientific community, we will make the data readily accessible to researchers via a user-friendly interface. !