Imaging System for Array Tomography

描述(由申请者提供)：我们申请资金购买一台荧光显微镜，配备灵敏的电荷耦合器件和电动平台，用于阵列断层扫描，以高分辨率确定多种蛋白质在脑组织中的位置。阵列断层成像是一种新的方法，它克服了传统免疫组织化学方法的两大局限性：z方向分辨率低和无法在单个样本中成像大量蛋白质。通过将组织嵌入聚合物中，然后将组织切片成一系列厚度约为100 nm的切片，可以克服较差的z分辨率。较小的切片厚度导致的z分辨率比传统的荧光成像或共焦成像要好得多。第二个限制通过阵列断层扫描允许每部分检查大量表位的事实得到解决，因为抗体可以有效地从制备中剥离，并且可以应用新的抗体。我们建议使用阵列断层扫描来表征细胞、突触前突触、突触后密度和棘突，以及蛋白质在细胞体中的分布。初步实验已经确立了我们准备组织、切割薄片阵列以及对单个免疫染色切片成像的能力。然而，我们缺乏一种合适的显微镜，可以从连续切片中自动收集大量图像。哈佛医学院目前还没有合适的显微镜，可以在长时间内对切片阵列进行高分辨率成像。因此，我们要求提供资金，以购买适合阵列断层扫描的显微镜，供这项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.