Super-resolution deep tissue imaging of dendritic spines

树突棘的超分辨率深层组织成像

• 批准号: 9269018
• 负责人: Meng Cui
• 金额: $ 10.2万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269018
• 关键词: Acoustics; Action Potentials; Adopted; Adult; Animals; BRAIN initiative; Brain; Brain Stem; Brain imaging; Calcium; Calcium Signaling; Cerebellum; Communities; Computer software; Conflict (Psychology); Dendritic Spines; Development; Electrodes; Engineering; Feedback; Fluorescence; Generations; Goals; Health; Image; Imaging Techniques; Label; Lasers; Life; Light; Location; Measurement; Measures; Methods; Microscope; Microscopy; Motion; Mus; Neck; Neocortex; Nervous system structure; Neurons; Neurosciences; Nobel Prize; Optics; Pattern; Performance; Play; Research; Resolution; Scanning; Science; Signal Transduction; Speed; Spinal Cord; System; Techniques; Tissue imaging; Tissues; Work; Zebrafish; absorption; adaptive optics; base; brain tissue; calcium indicator; cranium; data acquisition; design; flexibility; image processing; imaging modality; improved; in vivo; in vivo imaging; light microscopy; meetings; neural circuit; neuronal cell body; non-invasive imaging; optogenetics; temporal measurement; two-photon; user-friendly

 DESCRIPTION (provided by applicant): High resolution deep tissue calcium imaging with large field of view wavefront correction Two-photon microscopy based calcium imaging allows in vivo observation of neuronal dynamics at high spatial and temporal resolutions. The latest development allows single action potential sensitivity and single dendritic spine resolution, which provides a powerful solution to investigate the function of neural circuits. However, such resolution and sensitivity can only be achieved for the upper ~400 µm of neocortex of adult mice. Most of the studies are still focused on layer 2/3 neurons. New methods are urgently needed to investigate the layer 5 and 6 neurons. The challenge of deep tissue imaging is not light absorption but the aberration and scattering that distort the optical wavefront and the laser focus. Despite the apparent randomness, optical wavefront distortion can in principle be completely canceled by proper wavefront correction. New methods have emerged to enable high resolution imaging in highly turbid tissue. In effect, the new generation of wavefront correction methods provides an optical tissue clearing that can work for in vivo imaging. However, the current state-of-the-art methods still have various constraints to meet all the requirements of common calcium imaging procedure. Ideally, we need methods that can work on behaving animals at flexible wavelength (0.93, 1.1, 1.3-1.4, 1.7 µm). The method should require no additional labels other than the calcium indicator. To have a turn-key solution, the method needs to be automatable. Here we propose a robust solution based on our previous development, which can meet all the requirements of common calcium imaging procedure. The major bottle neck of the state-of-the-art wavefront correction methods is the tradeoff between the correction field of view (FOV) and correction quality. Tiling has been employed in the past to form a larger FOV, which nevertheless slows down the imaging process. We propose a new method to fundamentally remove the tradeoff between FOV and quality to achieve high resolution calcium imaging at great depth without sacrificing speed and FOV. This development is useful for not only multiphoton microscopy but also the emerging wide field microscopy methods such as the light sheet microscopy and light field microscopy. Besides calcium imaging, the developed large FOV wavefront measurement and correction can also benefit deep tissue optogenetics, especially for patterned excitation. These systems will be developed collaboratively by the engineers at Purdue and the neurobiologists at NYU. The neurobiologists will advise the system design. Once completed, the developed system will be delivered to the NYU lab for applications on neuroscience studies, which provides feedback to the engineers to further optimize the system. The ultimate goal is to have a turn-key solution that can be easily adopted by neurobiologists. We will make the system design (optics, optomechanics, data acquisition system, control software) freely available to the neuroscience community to quickly disseminate these methods.

 描述（由适用提供）：高分辨率深层组织钙成像，具有较大的视场波饰校正两光子显微镜钙成像，可以在高空间和临时分辨率下进行体内观察神经元动力学。最新的发展允许单个动作势灵敏度和单一树突状脊柱分辨率，该分辨率为研究神经元电路的功能提供了强大的解决方案。但是，只能针对成年小鼠的新皮层的上部〜400 µm实现这种分辨率和灵敏度。大多数研究仍然集中在2/3层神经元上。迫切需要使用新方法来研究第5层和6层神经元。深层组织成像的挑战不是轻滥用，而是扭曲光波前和激光的差异和散射 重点。尽管有明显的随机性，但原则上可以通过适当的波前校正完全取消光波前变形。已经出现了新的方法，可以在高度浊度组织中进行高分辨率成像。实际上，新一代的波前校正方法提供了可以用于体内成像的光学组织清除。但是，当前的最新方法仍然具有各种限制，以满足常见钙成像程序的所有要求。理想情况下，我们需要可以在柔性波长（0.93、1.1、1.3-1.4、1.7 µm）下进行行为动物的方法。该方法应不需要除钙指标以外的其他标签。要具有交钥匙解决方案，该方法需要自动化。在这里，我们根据我们先前的开发提出了一个可靠的解决方案，该解决方案可以满足常见钙成像程序的所有要求。最先进的波前校正方法的主要瓶颈是校正场（FOV）和校正质量之间的权衡。过去，瓷砖已被用来形成更大的FOV，但仍减慢了成像过程。我们提出了一种新的方法，可以从根本上消除FOV与质量之间的权衡，以在不牺牲速度和FOV的情况下以极大的深度实现高分辨率钙成像。这种发展不仅可用于多光子显微镜，而且对新兴的宽场显微镜方法（如光片显微镜和光场显微镜）有用。除了钙成像外，开发的大型FOV波前测量和校正也可以使深层组织光遗传学受益，尤其是对于图案化的令人兴奋。这些系统将由普渡大学的工程师和纽约大学的神经生物学家协作开发。神经生物学家将为系统设计提供建议。完成后，开发的系统将交付到NYU实验室，以进行神经科学研究的应用，该应用程序为工程师提供了反馈，以进一步优化系统。最终的目标是拥有一个可以由神经生物学家轻松采用的交钥匙解决方案。我们将免费提供系统设计（光学机械，数据采集系统，控制软件），可供神经科学社区迅速传播这些方法。