Ultrafast high-contrast voltage imaging in freely moving animals

摘要 我们建议开发一种靶向照明共聚焦（TICO）显微镜， 大脑中的大规模电压成像。这种显微镜将基于两种 关键战略。第一种策略是基于线扫描的高速共焦显微镜。的 共焦显微镜的好处是通过使用狭缝 侦测我们将补充这种背景拒绝与额外的战略，有针对性的 照明。通过这种方式，我们将仅将激发光限制在预先选定的感兴趣区域 (e.g.,细胞体或树突），从而最小化第一次成像中离焦背景的产生。 地方这些战略将通过使用双面扫描机制来实施 这将使我们能够执行快速扫描，以充分利用超快的吞吐量， 最先进的CMOS相机的低噪声能力。通过结合互补的 共焦检测和有针对性的照明的优势，我们希望显着提高 对比度和信噪比我们的电压成像相对于目前的方法基于相机。 我们的具体目标是建立两个双区域TICO显微镜，一个适用于头部固定 动物成像，另一个适用于使用微型摄像机进行自由移动的动物成像。 显微镜物镜由柔性纤维束耦合。确定TICO的可行性 在自由活动的动物身上使用显微镜将是一个重大突破， 为今后研究自然界中电压活动与行为之间的关系奠定了基础。 条件对自由移动的动物进行成像的能力将是实现这一目标不可或缺的。

ABSTRACT We propose to develop a targeted illumination confocal (TICO) microscope to enable high speed, large-scale voltage imaging in the brain. This microscope will be based on the combination of two key strategies. The first strategy is high-speed confocal microscopy based on line scanning. The benefit of confocal microscopy is that out-of-focus background is largely rejected by the use of slit detection. We will supplement this background rejection with the additional strategy of targeted illumination. In this way, we will confine our excitation light only to pre-selected regions of interests (e.g., cell bodies or dendrites), minimizing the generation of out-of-focus background in the first place. These strategies will be implemented with the use of a double-sided scanning mechanism that will enable us to perform fast scanning to fully exploit the ultrafast throughput and remarkably low noise capabilities of state-of-the-art CMOS cameras. By combining the complementary advantages of confocal detection and targeted illumination, we expect to significantly improve the contrast and SNR of our voltage imaging relative to the current methods based on cameras. Our specific aims are to build two dual-region TICO microscopes, one adapted for head-fixed animal imaging, and the other adapted for freely moving animal imaging using a miniature microscope objective coupled by a flexible fiber bundle. Establishing the feasibility of TICO microscopy in freely moving animals would constitute a major breakthrough and will lay the groundwork for future research into the relations between voltage activity and behavior in natural conditions. The ability to image freely moving animals will be indispensable for this goal.