TIRF Microscope with laser illumination and two EMCCD cameras

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Our studies seek to understand how the spatial organization and assembly dynamics of plasma membrane signaling pathways control T cell activation and functions. The research is based on our findings that plasma membrane proteins are localized in nanometer-sized domains. Our studies revealed that T cell activation is dynamically controlled by the spatial relationships of distinct nanodomains, the assembly of signaling complexes within them and translocation of signaling molecules between them. We study the underlying molecular mechanisms using multidisciplinary strategies that combine single molecule microscopy (i.e. single particle tracking and super-resolution) with biochemical, biophysical, and cell biological approaches. Specifically, we are interested in T cell receptor (TCR) and the inhibitory program cell death protein (PD-1) pathways during T cell activation and anti-tumor responses. Our ultimate goal is to identify novel control principles in T cell signaling pathways and use them to modify T cell responses for future immunotherapies.We request an advanced Total Internal Reflection (TIRF) microscope, which is essential to our newly established laboratory at the Albert-Ludwigs-Universität Freiburg and for realizing our current and future research goals. The requested microscope has the following capabilities: (i) Total Internal Reflection Microscopy, where illumination is limited to the contact site between cells and glass surfaces. (ii) Single molecule detection for super resolution imaging and particle tracking to analyze protein distributions and dynamics. Images are acquired with two EMCCD cameras for maximal sensitivity and speed. (iii) Localized laser control for photo-manipulation to measure protein dynamics and dispersion. Four high powered lasers covering the illumination wavelengths most significant for our research and method development. (iv) Widefield illumination for fast acquisition and calcium analyses. (v) Environmental control for live cell imaging.