A Multi-user Super Resolution Microscope for Developmental Biology

用于发育生物学的多用户超分辨率显微镜

基本信息

批准号：
7836356
负责人：
Samuel M Kunes
金额：
$ 104.95万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-11 至 2012-09-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7836356
关键词：
Afferent Neurons Area Brain Cells Cerebral cortex Chromatin Color Communities Complex Confocal Microscopy Cues Development Developmental Biology Developmental Process Electron Microscopy Erinaceidae Event Histocompatibility Image Knowledge Laboratories Laser Scanning Confocal Microscopy Lighting Maintenance Mango - dietary Maps Microscope Microscopy Molecular Molecular Probes Morphology Movement Mus Neural tube Neuromuscular Junction Neurons Nodal Pathway interactions Pattern Presynaptic Terminals Principal Investigator Protein Family Request for Proposals Resolution Role Signal Pathway Specimen Strabismus Structure Structure of parasympathetic ganglion Synapses Technology Time base developmental plasticity imaging modality instrument light microscopy long term memory morphogens nanoscale neural circuit vomeronasal organ zebrafish development

项目摘要

DESCRIPTION (provided by applicant): Understanding the molecular basis of developmental mechanisms ultimately requires precise knowledge of events in space and time. While the clearest view of subcellular organization has come from electron microscopy, its utility in understanding mechanism is burdened by the difficulty of mapping molecule to structure. Recent advances now offer us nanometer-scale resolution through technologies like STED, PALM/STORM and SIM that bring the resolution of light microscopy below the Abbe limit. This proposal requests support for the purchase of a Zeiss LSM710 PAL-M/SIM microscope. This instrument offers three imaging modalities; laser scanning confocal microscopy, Photo Activated Localization Microscopy (PAL-M), and Structured Illumination Microscopy (SIM). With this combination, one can obtain in a single specimen a broad 3D perspective by confocal microscopy and, in selected regions, sub-diffraction limit resolution with SIM and PAL-M. In combination with a wide array of multi-color fluorescent molecular probes, these are powerful technologies that open vast new areas of inquiry. In this proposal, the Principal Investigator Sam Kunes and five group leaders describe developmental biology projects that implement this technology. Developmental processes are a particularly suitable subject for super-resolution microscopy since they are governed and fine-tuned by spatial cues, many of which act in the sub-diffraction limited range. Three groups, Kunes, Lichtman and Dulac, propose to study the development of the ultrastructure of synaptic connections. Kunes will investigate how a developmental pathway is re-deployed to control long-term memory control axonal and dendritic branching, synapse number, and synapse morphology in the mature fruitfly brain. Lichtman plans to decipher the development of complex patterns of axonal circuitry and synaptic connectivity at the neuromuscular junction, and between neurons in the parasympathetic ganglia and cerebral cortex, using Brainbow mice. The Dulac laboratory will investigate the role of the Major Histocompatability M10 family of proteins in the developmental plasticity of vomeronasal organ sensory neuron axon terminals. Two groups, McMahon and Schier, propose to visualize morphogen movement at high resolution; McMahon will focus on Sonic Hedgehog, as it patterns the ventral neural tube during CNS development, while Schier proposes to image the morphogen Squint and its co-localization with other components of the Nodal signaling pathway during Zebrafish development. The Mango laboratory seeks to understand how the packaging of chromatin is modulated during development, in order to understand the transition from developmental plasticity to cell fate commitment. The instrument will be incorporated into a well-established imaging facility, where it will receive full-time technical support and oversight, support for maintenance and managed usage, thus providing long term availability to the co-PI group and the broader community of microscopists.

描述（由申请人提供）：了解发展机制的分子基础最终需要精确的时空事件知识。虽然亚细胞组织的最清晰视图来自电子显微镜，但其在理解机制方面的效用是由将分子映射到结构的困难所负担。现在的进步现在通过Sted，Palm/Storm和SIM等技术为我们提供了纳米尺度的分辨率，这些技术将光显微镜的分辨率低于ABBE限制。该建议要求支持购买Zeiss LSM710 PAL-M/SIM显微镜。该乐器提供三种成像方式；激光扫描共聚焦显微镜，照片激活的定位显微镜（PAL-M）和结构化照明显微镜（SIM）。通过这种组合，可以通过共聚焦显微镜在单个样品中获得广泛的3D透视图，并在选定的区域中使用SIM和PAL-M分辨率分辨率分辨率。结合各种多色荧光分子探针，这些都是强大的技术，可以打开广泛的新调查领域。在该提案中，首席研究员Sam Kunes和五个小组领导人描述了实施该技术的发展生物学项目。发育过程是超分辨率显微镜的特别适合的主题，因为它们是由空间提示控制和微调的，其中许多作用于子划分有限范围。库尼斯（Kunes），利希特曼（Lichtman）和杜拉克（Dulac）提议研究突触连接的超微结构的发展。库恩斯将研究如何重新启动发育途径，以控制成熟果蝇大脑中的长期记忆控制轴突和树突分支，突触数和突触形态。利希特曼计划使用Brainbow小鼠在神经肌肉连接处的轴突回路和突触连通性的复杂模式的发展，以及副交感神经神经节和大脑皮层的神经元之间的发展。 DULAC实验室将研究主要的组织相容性M10蛋白质家族在呕吐器官感觉神经元轴突末端的发育可塑性中的作用。 McMahon和Schier两组提议以高分辨率可视化形态学运动。 McMahon将重点放在Sonic刺猬上，因为它在CNS发育过程中对腹神经管进行了模式，而Schier则建议在斑马鱼发育过程中对形态学斜视及其与节点信号通路的其他组件进行成像。芒果实验室试图了解如何在发育过程中调节染色质的包装，以了解从发育可塑性到细胞命运承诺的过渡。该仪器将被纳入一个完善的成像设施中，在那里它将获得全职技术支持和监督，对维护和托管使用的支持，从而为Co-Pi集团和更广泛的显微镜研究员提供长期可用性。