Optimization of multiphoton microscopy for large scale activity mapping in adult zebrafish

成年斑马鱼大规模活动绘图的多光子显微镜优化

• 批准号: 9405203
• 负责人: JOSEPH R. FETCHO
• 金额: $ 75.42万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9405203
• 关键词: Adoption; Adult; Age; Air; Animal Model; Animals; Area; Behavior; Biological; Brain; Brain region; CRISPR/Cas technology; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Custom; Development; Embryo; Evaluation; Exposure to; Farming environment; Fiber; Fishes; Fluorescence Microscopy; Functional Imaging; Future; Generations; Goals; Image; Imaging technology; Individual; Industrialization; Interdisciplinary Study; Label; Larva; Lasers; Life; Light; Membrane; Microscope; Microscopy; Modeling; Monitor; Mus; Neurobiology; Neurons; Optics; Participant; Penetration; Phenotype; Photons; Physiologic pulse; Process; Rattus; Reagent; Research; Resolution; Resource Sharing; Sampling; Scanning; Signal Transduction; Site; Source; Speed; Structure; Technology; Testing; Time; TimeLine; Transgenic Organisms; Work; Zebrafish; adaptive optics; calcium indicator; commercialization; cranium; design; experience; fluorophore; imaging system; improved; in vivo; industry partner; innovation; light weight; microscopic imaging; multiphoton imaging; new technology; noradrenergic; novel; novel strategies; photonics; programs; technological innovation; technology development; tool; two-photon

Vertebrate behaviors emerge from interactions of neurons across the brain, but the tools for revealing neuronal structure and function at the cellular level in living animals access only small portions of the brain. We must move toward access to structure and function anywhere in the brain of individual adult, behaving animals. In vivo three photon (3P) microscopy, a recent, but proven, technology allows optical access to deeper structures than ever before in intact mammalian brains, but much optimization remains to catalyze its wider adoption. The plan of this project is to extend the reach of 3P microscopy both within brains and through the scientific community by developing new technology and proving its worth for imaging structure and function anywhere in the brain of adult zebrafish – a powerful vertebrate model. This project will extend the depth, speed and regional extent of imaging with 2P and 3P through a combination of technological improvements. Imaging depth will be enhanced by the development of a novel dual adaptive optics approach to correct optical aberrations that combines conjugate and standard adaptive optics to allow deep imaging through the skull with near diffraction limited resolution and improved signal. To enhance the breadth and speed of imaging, a novel approach will be developed with a light-weight, small, tandem piezo-fiber scan engine and a large field of view with the ability to raster scan any 2 to 4 sub-regions in the field. By determining and then applying optimum laser repetition rates and the best order of the nonlinear excitation as a function of depth, the number of neurons will be increased that can be imaged and reduce light exposure to improve longer term, repeated imaging through life. While the innovations will be useful for many animal models, they will be tested by imaging newly generated transgenic zebrafish lines made with CRISPR technology, as well as other established lines. The lines label neurons of different transmitter phenotype with membrane targeted fluorophores for structural imaging, or genetically encoded calcium indicators (gCaMPf or s) for functional imaging. The goal on the biological front is to provide the tools to allow the unique ability to image neuronal structure and function anywhere in the brain of an intact individual vertebrate at any time during its life, from embryo into adulthood.

脊椎动物行为来自整个大脑神经元的相互作用，但揭示的工具 活动物中细胞水平的神经元结构和功能仅进入大脑的一小部分。我们 必须努力进入单个成年人的大脑中的任何地方，表现动物。 在体内三photoson（3p）显微镜，最近但经过证实的技术允许光学访问更深层次 在完整的哺乳动物大脑中，结构比以往任何时候 采用。该项目的计划是在大脑内和通过 科学界通过开发新技术并证明其对成像结构和功能的价值 成人斑马鱼大脑中的任何地方 - 强大的脊椎动物模型。该项目将扩展深度， 通过技术改进组合，具有2p和3p的成像速度和区域范围。 通过开发一种新型的双重自适应光学方法来纠正光学的成像深度将得到增强 结合结合和标准自适应光学元件的畸变，可以通过头骨进行深层成像 接近衍射有限的分辨率和改进的信号。为了增强成像的广度和速度，一本小说 将使用轻质，小的，串联的压电纤维扫描引擎和大型视野开发方法 具有栅格扫描该场中任何2至4个子区域的能力。通过确定然后应用最佳 激光重复率和非线性兴奋的最佳顺序作为深度的函数，数量 神经元将增加，可以成像并减少光照暴露以改善长期，重复 一生成像。虽然创新对许多动物模型都有用，但它们将通过 成像用CRISPR技术制成的新产生的转基因斑马鱼线以及其他 建立的线条。具有膜靶向的不同发射器表型的线条标记神经元 用于结构成像的荧光团，或用于功能性的遗传编码钙指标（GCAMPF或S） 成像。生物方面的目标是提供工具，以允许独特的神经元能力 完整个体脊椎动物的大脑中的任何地方的结构和功能在其生命中的任何时候，从 胚胎成年。