Technologies for real-time molecular and cellular imaging in live animals

神经科学的目标是了解神经系统如何在分子、细胞和网络水平上自我组织，将信号处理成信息。因此，以活动物及其完整的神经系统为实验范式是绝对必要的。然而，由于我们在适当水平上观察活动物完整的神经系统的能力有限，无论是分子、细胞还是系统，这项研究工作都受到了极大的阻碍。我的团队与一个神经生物学家团队密切合作，用基于光子学的技术来解决这个缺乏适当工具的问题。这是一个漫长的过程，其根源在于展示了如何使用明智地选择的物理现象来提取以前无法获得的信息，或者如何将光束整形和光学设计中的几个概念整合在一起，从而产生具有非常理想的特性的新型设备来研究活的神经系统。NSERC发现基金的目的是支持概念验证计划，使物理科学和工程专业的学生能够开发这样的工具。 这项研究计划的总体愿景是开发新的无标记成像方法来在分子、细胞和系统水平上评估神经系统的结构和功能，并开发必要的技术来克服与活体动物成像相关的许多挑战。为此，我们解决两个具体问题(动物运动和脂膜顺序)，并通过需要深入了解光-物质相互作用和光学设计的项目来解决它们。 首先，我们将开发一种适用于所有共焦和多光子成像系统的动物位置实时测量系统。在二次照明路径中使用特殊透镜将允许检测组织的表面，然后该表面将被用于调整物镜的位置以实现闭环稳定，或者用于测量和标记帧的位置并在动物运动的情况下重建3D图像。 其次，我们建议使用偏振敏感的相干拉曼成像来成像髓鞘等脂膜的分子有序性。相干拉曼过程多年来一直被用于成像髓鞘(白质)的脂类，并可以产生神经系统中白质的内源性图像。在这里，我们将利用相干拉曼过程的偏振敏感性来提取髓鞘内脂类的分子顺序，并在不同偏振下拍摄一组图像。由于一种新的策略来补偿显微镜光学部件引入的偏振失真，这将成为可能。有了这个创新的系统，我们将能够快速而稳健地进行这些分子顺序测量，以提取以前无法获得的信息。

The goal of neuroscience is to understand how the nervous system organizes itself at molecular, cellular and network levels to process signals into information. Experimental paradigms with live animals and their intact nervous system are therefore absolutely necessary. However, this research effort is greatly impeded by our limited ability to observe the intact nervous system in a live animal at the appropriate level, be it molecular, cellular or systemic. My group works in close collaboration with a team of neurobiologists to address this lack of appropriate tools with photonics-based technology. This is a lengthy process that has its origin in demonstrating how judiciously chosen physical phenomena can be used to extract previously unobtainable information, or how the integration of several notions from beam shaping and optical design can lead to novel devices with highly desirable properties to study the live nervous system. The purpose of this NSERC Discovery grant is to feed the proof-of-concept program that makes possible the development of such tools with students from physical sciences and engineering. The overall vision of this research program is to develop novel label-free imaging methods to assess the structure and function of the nervous system at molecular, cellular and systemic levels, and develop the necessary technologies to overcome the many challenges associated with their use for live animal imaging. To do so, we tackle two specific problems (animal movement and lipid membrane order) and address them with projects that require an intimate knowledge of light-matter interactions and optical design. First, we will develop a system for real-time measurement of animal position that is adaptable to all confocal and multiphoton imaging systems. The use of a special lens in a secondary illumination path will allow the detection of the surface of the tissue, which will then be used either to adjust the position of the objective for closed-loop stabilization, or to measure and label the position of the frames and reconstruct a 3D image despite animal movement. Second, we propose to use polarization-sensitive coherent Raman imaging to image the molecular order of lipid membranes such as myelin.  The coherent Raman process has been used for years to image the lipids of myelin (white matter), and can yield endogenous images of the white matter in the nervous system.  Here we will turn to our advantage the polarization-sensitivity of the coherent Raman process to extract the molecular order of lipids within myelin with a set of images at different polarizations.  This will be made possible thanks to a novel strategy to compensate polarization distortion introduced by the optical components of the microscope. With this innovative system, we will be able to perform these molecular order measurements rapidly and robustly to extract previously unobtainable information.