Rapid super-resolution optical imaging of immunological synapse formation initiated by holographic optical tweezers

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This research proposal aims at developing a fast, super-resolving structured illumination microscope (SR-SIM) to resolve the structure and dynamics of signalling microclusters in living T cells. This is accomplished by implementing the SR-SIM image reconstruction process on fast parallel-processing graphics processor platforms to enable the imaging and displaying of reconstructed super-resolved images of living cells at video rate. The system will further integrate holographic optical tweezers that enable the manipulation (immobilization) of living, non-adherent immune cells within the focal plane of the microscope, and to initiate immunological synapse formation between living immune cells. To permit the three-dimensional reconstruction of images of living immune cells, the rapid acquisition of images from multiple image planes simultaneously, as well as compensation of spherical aberrations induced by the microscope optics will be included in the final optical system. The performance of this system will then be utilized to image an immunologically highly relevant targt, i.e. the vesicular trafficking of signalling molecules within T cells. The high temporal and spatial resoluton will enable us to determine if signalling molecules, such as TCR, CD4, and Lck are transported in the same or different vesicles. This system will allow us to image docking and fusion of individual vesicles delivered to the T cell plasma membrane, and it wil enable us to determine how vesicle transfer and clustering change between resting and activated T cells. The ability to control the position and orientation of non-adherent cells at will by optical tweezers, as well as to perform high-speed volumetric imaging will ultimately also enable us to determine the vesicular trafficking routes, rates, and degrees of clustering between interacting immune cells.