Super-resolution deep tissue imaging of dendritic spines

 描述(申请人提供)：具有大视场波前校正的高分辨率深层组织钙成像基于双光子显微镜的钙成像可以在高空间和时间分辨率下活体观察神经元动力学。最新的发展允许单动作电位灵敏度和单树突棘分辨，这为研究神经电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质上部~400微米处达到。大多数研究仍集中在2/3层神经元上。迫切需要新的方法来研究5层和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 全神贯注。尽管存在明显的随机性，但光学波前失真原则上可以通过适当的波前校正来完全消除。出现了能够在高度混浊的组织中进行高分辨率成像的新方法。实际上，新一代波前校正方法提供了一种可用于活体成像的光学组织清晰度。然而，目前最先进的方法仍然有各种限制，以满足常见的钙成像程序的所有要求。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微米)下工作的动物的方法。除钙指示剂外，该方法不需要额外的标签。为了有一个交钥匙解决方案，该方法需要是自动化的。在此，我们提出了一种健壮的解决方案，它可以满足普通钙质成像程序的所有要求。现有波前校正方法的主要瓶颈是校正视场(FOV)和校正质量之间的权衡。在过去，平铺被用来形成更大的视场，但这会减慢成像过程。我们提出了一种新的方法，从根本上消除了视场和质量之间的权衡，在不牺牲速度和视场的情况下，实现了大深度的高分辨率钙成像。这一发展不仅对多光子显微镜是有用的，而且对新兴的广视场显微镜方法，如光片显微镜和光场显微镜也是有用的。除了钙成像，开发的大视场波前测量和校正也有助于深层组织光遗传学，特别是图案化激发。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。一旦完成，开发的系统将被交付给纽约大学实验室用于神经科学研究，该实验室向工程师提供反馈，以进一步优化系统。最终目标是拥有一种易于神经生物学家采用的交钥匙解决方案。我们将向神经科学界免费提供系统设计(光学、光学机械、数据采集系统、控制软件)，以迅速传播这些方法。

 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.