Imaging System for Array Tomography

描述（由申请人提供）：我们请求资金购买一台配备灵敏CCD和阵列断层扫描电动载物台的荧光显微镜，以高分辨率确定脑组织中多种蛋白质的位置。阵列断层扫描是一种新的方法，克服了传统免疫组织化学方法的两个主要局限性：z方向的分辨率差和无法在单个样品中成像大量蛋白质。通过将组织包埋在聚合物中，然后将其切成一系列厚度约为100 nm的切片来克服z分辨率差。小的切片厚度导致z分辨率比常规荧光成像或共焦成像所能实现的要好得多。第二个限制是解决了阵列断层扫描允许大量的表位进行检查每个部分，因为抗体可以有效地从制备和新的抗体可以应用。我们建议使用阵列断层扫描来表征细胞，突触前的终扣，突触后的密度和棘，以及蛋白质在细胞体中的分布。初步的实验已经建立了我们的能力，准备组织，薄切片的切割阵列，和图像个人免疫染色切片。然而，我们缺乏一个合适的显微镜，将允许自动收集大量的图像从连续切片。目前在哈佛医学院还没有合适的显微镜，可以在很长的时间内以高分辨率成像切片阵列。因此，我们要求提供资金，以购买一台适合阵列断层扫描的显微镜，供本提案的主要用户使用。

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.