High resolution Imaging: 3-D EM reconstrucfion and STORM microscopy

项目总结(见说明)： 目前对大脑回路的详细环境数据的障碍主要是技术上的。在这个核心中，我们将建立两个强大的成像模式，提供有关支配一类皮质神经元的突触的数量和类型的详细信息，此外，还提供神经元支配的所有目标细胞的完整列表。两种成像方式之一是由核心领导者之一发明的被称为STOCHASFIC光学重建显微镜(STORM)的超分辨率荧光成像方法。这种方法提供了纳米级的成像分辨率，具有识别特定神经元和突触类型所需的分子特异性。在核心部分，我们将开发染色和成像功能，以实现亚衍射极限分辨率的神经元和相关突触的可视化。我们的进一步目标是使成像过程自动化，以允许常规地重建神经元的整个突触分布，特别是其突触前输入。第二种成像方式是连续电子显微镜。 将在核心支持一种新的电子显微镜方法，允许在大量脑组织中进行高分辨率神经电路重建。这种方法使用了一种新型的自动切片机，将大脑的超薄切片放在磁带上，并使用自动二次电子检测扫描电子显微镜对这些切片进行成像。作为STORM设备的补充，这一系列EM设备将致力于重建特定神经元的整个突触后靶场。核心的工作人员不仅将使这些成像设施可供三个PI实验室的研究人员使用，还将与这些实验室的研究人员密切合作，开发项目目标所需的特定成像能力

PROJECT SUMMARY (See instructions): The present impediments to detailed connetomic data of brain circuitry are largely technical. In this core we will set up two powerful imaging modalities that provide detailed information about the number and type of synapses that innervate a class of cortical neurons and in addition, provide the full listing of all the targets cells a neuron innervates. One of the two imaging modalities is a super-resolution fluorescence imaging method termed STochasfic Optical Reconstruction Microscopy (STORM) invented by one of the core leaders. This method offers nanometer-scale imaging resolution with molecular specificity needed to identify specific neuronal and synapse types. In the core, we will develop staining and imaging capabilities to allow visualization of neurons and associated synapses with sub-diffraction-limit resolution. We further aim at automating the imaging process to allow the entire synapse distribution of a neuron, in particular its presynaptic input, to be routinely reconstructed. The second imaging modality is serial electron microscopy. A new approach to electron microscopy will be supported in the core that allows high resolution reconstruction of neural circuits in large volumes of brain tissue. The approach uses a novel automated microtome that puts ultrathin sections of brain on tape and an automated secondary-electron-detecting scanning electron microscope to image these sections. Complementary to the STORM facility, this serial EM facility will aim at reconstruction of the entire postsynaptic target field of specific neurons. Staff members in the core will not only make these imaging facilities available to researchers in the three Pi's lab, but also work closely with researchers in these labs to develop specific imaging capabilities needed for the project goals