Optimization of 3-photon microscopy for Large Scale Recording in Mouse Brain

用于小鼠大脑大规模记录的三光子显微镜优化

• 批准号: 9130300
• 负责人: CHRIS XU
• 金额: $ 48.32万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9130300
• 关键词: Academia; Area; Biomedical Research; Brain; Brain imaging; Collaborations; Collection; Communities; Dendrites; Deposition; Development; Device or Instrument Development; Elements; Feedback; Fiber; Fluorescence Microscopy; Generations; Genetic Engineering; Goals; Green Fluorescent Proteins; Head; Health; Image; Imaging Device; Individual; Industry; Interdisciplinary Study; Lasers; Lead; Microscope; Microscopy; Mus; Neurobiology; Neurons; Neurosciences; Noise; Optics; Participant; Penetration; Performance; Photons; Physiologic pulse; Procedures; Process; Proteins; Research; Research Personnel; Resolution; Signal Transduction; Source; Speed; System; Technology; Testing; Tissues; Universities; Work; adaptive optics; awake; base; biological research; brain tissue; commercialization; design; experience; fluorophore; improved; in vivo; industry partner; instrument; lens; meetings; performance tests; programs; research and development; tool; two-photon; virtual reality

 DESCRIPTION (provided by applicant): The goal of this research program is to optimize three-photon fluorescence microscopy (3PM) for large scale, noninvasive, volumetric imaging of neuronal activity. To leverage the superb performance of green-fluorescent protein based genetically engineered Ca-probes (e.g., GCaMPs), 3PM at the 1300-nm spectral window will be developed, which not only preserves the tissue penetration capability of 3PM at the longer excitation wavelength but also enables a wide variety of blue and green fluorophores, including a number of fluorescent proteins and Ca-indicators, to be excitable via three-photon excitation. To improve the signal-to-noise ratio (SNR) so that a practical frame rate can be achieved for imaging dynamic brain activity even at a penetration depth of 1.1 mm or beyond, new objective lenses will be designed and fabricated that will collect the signal efficiently at depth. In additin, the lens design will also support convenient integration with adaptive optics (AO), with the goal of making AO a routine imaging tool in a neuroscience lab. To improve both SNR and spatial resolution, AO in 3PM at 1300 nm will be employed. The impact of AO for increasing signal generation is significantly higher for 3PM than 2PM because of the higher order nonlinear process. The impact of AO is also expected to increase with increasing imaging depth. The aim is to achieve close to diffraction limited spatial resolution for 3PM at 1300 nm, which will be sufficient to resolve individual dendrite. A strong interdisciplinary research team has been assembled, including participants from both industry and academia, to perform the research and development. The successful completion of this program will have a broad impact on neuroscience where high-resolution, high speed imaging deep within an intact mouse brain is required.

 描述（由申请人提供）：该研究计划的目标是优化三光子荧光显微镜（3PM），用于神经元活动的大规模、无创、体积成像，以利用基于绿色荧光蛋白的基因工程 Ca 的卓越性能。 -将开发1300 nm光谱窗口的3PM探针（例如GCaMP），这不仅保留了3PM在1300 nm光谱窗口的组织穿透能力更长的激发波长，而且还可以通过三光子激发来激发各种蓝色和绿色荧光团，包括许多荧光蛋白和 Ca 指示剂，从而提高信噪比 (SNR)。即使在 1.1 毫米或以上的穿透深度下，也可以实现对动态大脑活动进行成像的实用帧速率，将设计和制造新的物镜，以有效地收集深度信号。此外，镜头设计也将支持。与自适应光学 (AO) 方便地集成，目标是使 AO 成为神经科学实验室的常规成像工具 为了提高 SNR 和空间分辨率，将采用 3PM 1300 nm 的 AO 来增加信号生成。 3PM 的空间分辨率明显高于 2PM，因为高阶非线性过程预计 AO 的影响也会随着成像深度的增加而增加，目的是在 3PM 下实现接近衍射极限的空间分辨率。 1300 nm，足以解析单个树突，已经组建了一个强大的跨学科研究团队，包括来自工业界和学术界的参与者，以进行该项目的成功完成将对神经科学产生广泛的影响。需要在完整的小鼠大脑深处进行高分辨率、高速成像。