Three of the investigators on this application (Storrie at the University of Arkansas for Medical Sciences, Brooker and Rosen at Johns Hopkins University and Ben Gurion University) have been collaborating for the last 7 years on the vision that application of holography to fluorescence microscopy could simplify 3D microscopy and also capitalize on the super-resolution capabilities afforded by holography. This work, supported by NSF, NIH SBIR, and NIST grants hasnow yielded a 3-dimensional, fluorescence microscopic technique with super-resolving properties. It is based upon a new holography concept called FINCH, developed by two of the investigators (Rosen and Brooker). FINCH is a holographic technique in which the incoherent fluorescence microscope emission is imaged with a simple setup consisting of a spatial light modulator inserted into the light path of a standard fluorescence microscope stand, an attached digital camera, and a non-laser, externally shuttered light source. Recently the FINCH technique was found to have inherent super-resolving properties. This system has been developed to the stage of a bench-top hardware and software prototype. In this proposal, the network of 4 investigators at three universities including Vladimir Lupashin at University of Arkansas for Medical Sciences will participate in three aspects of the implementation, research use and further improvement ofthe FINCH super-resolution microscope technology. 1. At Johns Hopkins University (JHU), Brooker and Rosen will implement a stand-alone FINCH microscope, supporting algorithms and image acquisition and hologram reconstruction software to be installed at UAMS during the middle of grant Year 2. 2. At University of Arkansas for Medical Sciences (UAMS), Storrie and Lupashin will utilize the FINCH microscope to advance innovative research in membrane trafficking and Golgi organization that would not otherwise be possible. In that work, they will also compare FINCH microscope performance with other super-resolution techniques. 3. At Ben Gurion University (BGU), Rosen will continue to develop advanced algorithms and concepts based upon FINCH to improve performance and resolving power.
We propose the implementation and application of the novel approach of super-resolution holography to the study of Golgi apparatus function in live cells. The Golgi apparatus is the central organelle within the secretory pathway of human cells. How the structure and function of this organelle is regulated is central to the health of cells and hence to the health humans. Defects in Golgi associated Rab protein function affect vision (glaucoma), neurodegenerative disease, Chlamydia! infection, viral entry and aging.
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