A Multi-user Super Resolution Microscope for Developmental Biology

描述（由申请人提供）：了解发育机制的分子基础最终需要对空间和时间事件的精确了解。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受到分子到结构映射的困难的影响。最近的进展现在通过STED，PALM/STORM和SIM等技术为我们提供了纳米级的分辨率，这些技术使光学显微镜的分辨率低于阿贝极限。该提案要求支持购买蔡司LSM 710 PAL-M/SIM显微镜。该仪器提供三种成像模式;激光扫描共聚焦显微镜，光激活定位显微镜（PAL-M）和结构照明显微镜（SIM）。通过这种组合，可以在单个样品中通过共焦显微镜获得广泛的3D视角，并且在选定的区域中，使用SIM和PAL-M获得亚衍射极限分辨率。结合各种多色荧光分子探针，这些都是强大的技术，开辟了广阔的新领域的调查。在这份提案中，首席研究员Sam Kunes和五位小组负责人描述了实施这项技术的发育生物学项目。发育过程是一个特别适合的主题，因为它们是由空间线索，其中许多行为在子衍射限制范围内的治理和微调超分辨率显微镜。Kunes、Lichtman和迪拉克这三个研究小组建议研究突触连接超微结构的发育。Kunes将研究如何重新部署发育途径来控制成熟果蝇大脑中的长期记忆控制轴突和树突分支，突触数量和突触形态。Lichtman计划使用Brainbow小鼠来破译神经肌肉接头处以及副交感神经节和大脑皮层中的神经元之间的轴突回路和突触连接的复杂模式的发展。迪拉克实验室将研究主要组织相容性M10蛋白家族在犁鼻器感觉神经元轴突终末发育可塑性中的作用。McMahon和希耶两个小组提出以高分辨率可视化形态发生运动; McMahon将专注于Sonic Hedgehog，因为它在中枢神经系统发育期间形成腹侧神经管的图案，而希耶提出在斑马鱼发育期间成像形态发生斜视及其与Nodal信号通路的其他组分的共定位。Mango实验室试图了解在发育过程中染色质的包装是如何调节的，以了解从发育可塑性到细胞命运承诺的转变。该仪器将被纳入一个完善的成像设施，在那里它将得到全职的技术支持和监督，维护和管理使用的支持，从而提供长期可用性的共同PI组和更广泛的社区显微镜。

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.