Imaging System for Array Tomography

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DESCRIPTION (provided by applicant): We request funds to purchase a fluorescence microscope equipped with a sensitive CCD and a motorized stage for array tomography to determine with high resolution the location of multiple proteins in brain tissue. Array tomography is a new approach that overcomes two major limitations of conventional immunohistochemical approaches: the poor resolution in the z direction and the inability to image large numbers of proteins in a single sample. The poor z resolution is overcome by embedding tissue in a polymer that is then sectioned in a series of slices of about 100 nm in thickness. The small slice thickness results in a z resolution that is much better than can be achieved with conventional fluorescence imaging or confocal imaging. The second limitation is resolved by the fact that array tomography allows a large number of epitopes to be examined per section because antibodies can be efficiently stripped from the preparation and new antibodies can be applied. We propose to use array tomography to characterize cells, presynaptic boutons, postsynaptic densities and spines, and the distribution of proteins in the cell body. Preliminary experiments have established our ability to prepare tissue, cut arrays of thin sections, and image individual immunostained slices. However, we lack an appropriate microscope that will allow automated collection of a large number of images from serial sections. There is currently no suitable microscope at Harvard Medical School that is available for the long blocks of time necessary to image slice arrays at high-resolution. We are therefore requesting funds to purchase a microscope suitable for array tomography for the use of the major users on this proposal.