Super-resolution deep tissue imaging of dendritic spines

 描述（由申请人提供）：具有大视场波前校正的高分辨率深部组织钙成像基于双光子显微镜的钙成像允许以高空间和时间分辨率在体内观察神经元动力学。最新的发展允许单个动作电位灵敏度和单个树突棘分辨率，这为研究神经回路的功能提供了强大的解决方案。然而，这样的分辨率和灵敏度只能在成年小鼠的新皮层的上部~400 µm处实现。大多数研究仍然集中在2/3层神经元。迫切需要新的方法来研究第5层和第6层神经元。 深层组织成像的挑战不是光吸收，而是使光学波前和激光失真的像差和散射 专心点尽管存在明显的随机性，但光学波前畸变原则上可以通过适当的波前校正完全消除。已经出现了新的方法来实现在高度混浊的组织中的高分辨率成像。实际上，新一代波前校正方法提供了可以用于体内成像的光学组织清除。然而，目前最先进的方法仍然有各种限制，以满足所有的要求，共同的钙成像程序。理想情况下，我们需要能够在灵活的波长（0.93，1.1，1.3-1.4，1.7 μm）下对动物行为起作用的方法。除钙指示剂外，该方法不需要其他标签。要有一个交钥匙解决方案，该方法需要是可自动化的。在这里，我们提出了一个强大的解决方案的基础上，我们以前的发展，它可以满足所有的要求，常见的钙成像程序。 波前校正方法的主要瓶颈是校正视场和校正质量之间的权衡。过去已经采用平铺来形成更大的FOV，然而这减慢了成像过程。我们提出了一种新的方法，从根本上消除FOV和质量之间的权衡，以实现高分辨率的钙成像在很大的深度，而不牺牲速度和FOV。这一发展不仅对多光子显微术有用，而且对新兴的宽视场显微术方法如光片显微术和光场显微术也有用。除了钙成像之外，所开发的大FOV波前测量和校正也可以有益于深部组织光遗传学，特别是对于图案化激发。 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合作开发。神经生物学家将为系统设计提供建议。完成后，开发的系统将交付给纽约大学实验室用于神经科学研究，该实验室为工程师提供反馈，以进一步优化系统。最终目标是有一个交钥匙解决方案，可以很容易地被神经生物学家采用。我们将使系统设计（光学，光学机械，数据采集系统，控制软件）免费提供给神经科学界，以快速传播这些方法。

 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.