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.

PUBLIC HEALTH RELEVANCE: The proposal is to develop and optimize a new form of deep tissue imaging method that can enable in vivo high resolution imaging of mammalian brains at tissue depths that are currently unavailable.