Implantable 3D fluorescence imaging with high-speed, addressable laser scanning in moving mice

项目摘要 这项工作的目标是在植入式光纤耦合显微镜中使用微型扫描镜，实现对活动、行为正常的小鼠脑体积的高速光学荧光成像。现有的用于神经成像的植入式显微镜主要成像单个焦平面，或者可能使用速度较慢和/或降低光学分辨率的扫描机制来改变图像深度。基于微电子机械系统(MEMS)技术的扫描镜可以放置在光纤耦合显微镜的物镜附近，获得很高的扫描频率。基于有源压电材料的MEMS扫描仪可以快速引导激光穿过空间的所有三个轴。然后，利用超快激光激发的多光子效应，可以通过深度成像穿透进行荧光成像。我们的具体目标是：(1)展示高性能的MEMS反射镜，用于轴向和横向扫描，其速度和形状适合于脑成像；(2)将反射镜集成到植入式显微镜原型中，以实现细胞到亚细胞分辨率的3D成像；(3)收集运动小鼠的神经激活图像。较小的镜面质量和主动扫描仪控制将防止不需要的运动伪影，并提高图像体积内局部感兴趣区域的采样频率。这项工作的成功结束将为在3D大脑环境中较长时间内的神经元行为提供新的工具，这对于研究包括运动、社会行为和学习在内的神经功能是必要的。

Project Summary The goal of this work is to achieve high-speed optical fluorescence imaging of 3D brain volume in moving, behaving mice, using miniature scanning mirrors in an implantable, fiber-coupled microscope. Existing implantable microscopes for neural imaging primarily image a single focal plane or may alter image depth using scanning mechanisms that are slow and/or reduce the optical resolution. Scanning mirrors based on microelectromechanical system (MEMS) technology can be placed near the objective lens of fiber-coupled miniature microscopes, and achieve very high scanning frequencies. MEMS scanners based on active piezoelectric materials can rapidly steer laser light through all three axes in space. Fluorescence imaging can then be performed with deep imaging penetration using the multi-photon effect excited by an ultrafast laser. Our Specific Aims are to (1) demonstrate high-performance MEMS mirrors for axial and lateral scanning with speed and form factor appropriate for brain imaging; (2) integrate mirrors into a prototype implantable microscope, for 3D imaging at cellular-to-sub-cellular resolution; (3) collect images of neural activation from moving mice. Small mirror mass and active scanner control will prevent undesired motion artifacts and increase sampling frequencies in localized regions of interest within the image volume. The successful conclusion of this work would provide novel instruments for neuron behavior in the 3D brain environment over extended periods, necessary for the study of neural functions including locomotion, social behavior, and learning.