Zeiss Axioimager Z2 Imaging System for Array Tomography

用于阵列断层扫描的蔡司 Axioimager Z2 成像系统

• 批准号: 8050429
• 负责人: Sacha B Nelson
• 金额: $ 15.65万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-15 至 2013-03-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050429
• 关键词: Antibodies; Biological Assay; Cataloging; Catalogs; Cells; Communication; Confocal Microscopy; Cryoultramicrotomy; Development; Epilepsy; Excitatory Synapse; Functional disorder; Goals; Image; Imaging technology; Immunofluorescence Immunologic; Immunohistochemistry; Impairment; Inhibitory Synapse; Laboratory Organism; Mediating; Mental Retardation; Methods; Mission; Morphology; Nervous system structure; Neurologic; Neurons; Penetration; Peripheral Nervous System; Positioning Attribute; Proteins; Regulation; Research; Research Personnel; Resolution; Scanning Electron Microscopy; Site; Slide; Staining method; Stains; Structure; Synapses; System; Thick; Tissues; United States National Institutes of Health; Universities; autism spectrum disorder; brain tissue; fluorescence imaging; imaging modality; innovation; nervous system disorder; neuroregulation; novel; novel strategies; reconstruction; synaptogenesis; tomography

DESCRIPTION (provided by applicant): Synapses are specialized sites of cell-cell contact that mediate communication between neurons in the nervous system. It is now widely believed that aberrant development or function of either excitatory or inhibitory synapses contributes to neurological impairments such as mental retardation, autism spectrum disorders and epilepsy. To understand how synapse dysfunction underlies these neurological disorders, it is paramount that we discover how synapses form and function in the non-perturbed state. The NIH-sponsored research of the five investigators in the user group is specifically focused on this research goal: understanding how synaptic connections are formed, modified, and maintained in the nervous system of a variety of experimental organisms. Projects include: mechanisms of synapse formation in the central and peripheral nervous system, neuromodulation, and the regulation of intrinsic excitability. Array tomography is a novel imaging modality that represents a new approach to high resolution imaging of synaptic structure in intact nervous systems. The Zeiss Axio Imager Z2 fluorescence imaging system that we propose to purchase will allow this user group to study synaptic structure and function at an unprecedented resolution using an innovative sectioning and reconstruction strategy devised by Dr. Stephen Smith and colleagues at Stanford University. Traditional immunohistochemsitry using antibodies against synaptic markers on brain tissue sections yields poor resolution of synaptic puncta due to antibody penetration problems in relatively thick tissue sections (e.g. 255m) and limited resolution along the Z axis during imaging, even when employing confocal microscopy. Array tomography circumvents these issues by antibody staining of ultrathin cryosections (e.g. 70nm) of nervous system tissue. There are numerous additional benefits to utilizing array tomography over traditional immunohistochemistry including the possibility of obtaining ultrastructural information from the tissue using scanning electron microscopy after immunofluorescence imaging and, perhaps most importantly for our purposes, the ability to perform multiple rounds of antibody staining of the same tissue section. As the position of the tissue is fixed on the slide, repeated staining with different antibodies against synaptic proteins will allow cataloging of which synaptic components are present or absent at all of the synapses in a single neuron. All five investigators in the user group have reached the limit of what can be achieved in assaying synapse morphology and function with traditional immunofluorescence methods. Thus, it is critical to furthering the research mission of these groups that the array tomography imaging technology be available on the Brandeis campus. PUBLIC HEALTH RELEVANCE: Numerous studies now point to defects in synapse formation as a possible cause for neurological disorders such as autism, mental retardation, and epilepsy. One approach to understanding how aberrant synapse formation contributes to these widespread neurological impairments is to first investigate how synapses are formed, maintained, and function in the non-pathological state using light microscopy. Acquisition of the Zeiss Axioimager Z2 System for Array Tomography will allow for unprecedented high-resolution imaging of synapses from the nervous system of a variety of experimental organisms.

描述（由申请人提供）：突触是神经系统中细胞与细胞接触的特殊部位，介导神经元之间的交流。现在人们普遍认为，兴奋性或抑制性突触的异常发育或功能会导致神经系统损伤，如智力迟钝、自闭症谱系障碍和癫痫。为了理解突触功能障碍是如何导致这些神经系统疾病的，我们首先要发现突触在非扰动状态下是如何形成和功能的。美国国立卫生研究院赞助的用户组中的五名研究人员的研究特别关注于这个研究目标：了解突触连接如何在各种实验生物的神经系统中形成、修改和维持。项目包括：中枢和周围神经系统突触形成机制，神经调节和内在兴奋性的调节。阵列层析成像是一种新的成像方式，它代表了完整神经系统突触结构高分辨率成像的新方法。我们建议购买的蔡司Axio Imager Z2荧光成像系统将允许这个用户组以前所未有的分辨率研究突触结构和功能，使用由斯坦福大学的Stephen Smith博士和他的同事设计的创新切片和重建策略。传统的免疫组织化学使用针对脑组织切片上突触标记物的抗体，由于抗体在相对较厚的组织切片（例如255m）中的渗透问题，导致突触点的分辨率较差，并且在成像过程中沿Z轴的分辨率有限，即使使用共聚焦显微镜也是如此。阵列断层扫描通过对神经系统组织的超薄冷冻切片（例如70nm）进行抗体染色来解决这些问题。与传统的免疫组织化学相比，利用阵列断层扫描还有许多额外的好处，包括在免疫荧光成像后使用扫描电子显微镜从组织中获得超微结构信息的可能性，也许对我们的目的最重要的是，对同一组织切片进行多轮抗体染色的能力。当组织的位置固定在载玻片上时，用不同的针对突触蛋白的抗体反复染色，将允许对单个神经元中所有突触中存在或不存在的突触成分进行分类。用户组中的所有五名研究人员已经达到了传统免疫荧光方法在分析突触形态和功能方面所能达到的极限。因此，阵列层析成像技术在布兰迪斯大学校园的应用对于进一步推进这些小组的研究任务至关重要。

项目成果

A Mammalian enhancer trap resource for discovering and manipulating neuronal cell types.

• DOI: 10.7554/elife.13503
• 发表时间: 2016-03-21
• 期刊: eLife
• 影响因子: 7.7
• 作者: Shima Y;Sugino K;Hempel CM;Shima M;Taneja P;Bullis JB;Mehta S;Lois C;Nelson SB
• 通讯作者: Nelson SB