A multi-foci objective lens for large scale brain activity recording

摘要 该项目的目标是开发一种新的双焦点折反射物镜，它将允许大规模记录 活体内的神经回路。该目标将实现更快的大脑区体积成像，通过同时 脑组织浅层双光子(2P)成像和深层三光子(3P)成像 提高了收集效率。尽管三光子显微镜(3 PM)允许在无法到达的深度进行成像 通过双光子显微镜(2 PM)，用较低的激发脉冲产生较大的荧光信号 在浅层组织层成像时的能量。因此，对于跨大深度的快速成像而言，最佳 方法是对浅层使用2 PM，对深层同时使用3 PM。实施 这种方法不可避免地需要物镜产生两个焦面，这两个焦面被一个大的 轴向距离，同时仍保持高空间分辨率和大视场。同时下午2点和3点 不仅可以利用这两种模式的优点，而且还可以更快地进行体积成像 巨大的脑柱。我们将开发一种双焦点折反射(即折射和反射)透镜，基于 分离不同波长激发光的光路的思想。这款镜头将配备两个 对于2P(&lt；1100 nm)和3P(&gt；1200 nm)激发波长，焦平面轴向间隔约600微米。 该设计方法还将激励路径和收集路径分开，并允许独立 对发射的荧光进行有效收集的优化。双焦点物镜将收集荧光 通过非成像路径返回，这使得所提议的折反射物镜具有大量的集合 数字光圈和大的采集视场。收集效率约为5倍 商业上可用的物镜，当成像深度(&gt；1毫米)到小鼠的大脑时。提高产品质量 信号采集效率将在不增加激励功率的情况下立即提高帧速率， 在这些深度实现高分辨率、高速成像。我们将设计、编造和验证这部小说 它将与调焦镜头相结合，以实现对小鼠大脑更快的体积成像。 该计划的成功完成将立即实现跨 大范围的深度(~1.2毫米)，例如记录小鼠大脑不同层的神经元数量。 该计划开发的技术将在大量生物医学领域产生潜在影响 如神经科学、免疫学和癌症生物学。

Abstract The goal of this project is to develop a novel bifocal catadioptric objective that will allow large-scale recording of neural circuits in vivo. The objective will enable faster volumetric imaging of large brain regions by simultaneous two-photon (2P) imaging of shallow layers and three-photon (3P) imaging of deep layers of brain tissues with improved collection efficiency. Although three-photon microscopy (3PM) allows imaging at depths inaccessible by two photon microscopy (2PM), 2P excitation generates larger fluorescence signal with lower excitation pulse energy when imaging at shallow tissue layers. Therefore, for fast imaging across a large depth, the optimum approach is to use 2PM for the superficial layers and 3PM for the deeper regions simultaneously. Implementation of this approach inevitably requires the objective lens to generate two focal planes that are separated by a large axial distance while still maintaining high spatial resolution and large field of view. Simultaneous 2PM and 3PM will not only allow for utilization of the advantages of both modalities but also for faster volumetric imaging of large brain columns. We will develop a bifocal catadioptric (i.e., both refractive and reflective) lens based on the idea of separation the optical paths of the excitation light with different wavelengths. The lens will feature two focal planes separated axially by ~ 600 µm for the 2P (< 1100 nm) and 3P (>1200 nm) excitation wavelengths. The design approach also separates the excitation path and the collection path and allows independent optimization for efficient collection of the emitted fluorescence. The bifocal objective will collect fluorescence back through non-imaging pathways, which enables the proposed catadioptric objective to have a large collection numerical aperture and a large collection field of view. The collection efficiency is approximately 5x higher than the commercially available objective lenses when imaging deep (>1 mm) into the mouse brain. Improving the signal collection efficiency will immediately increase the frame rate without increasing the excitation power, enabling high-resolution, high-speed imaging at these depths. We will design, fabricate and validate the novel objective lens and will combine it with focus-tunable lenses to enable faster volumetric imaging of mouse brains. The successful completion of this program will immediately enable simultaneous 2P and 3P imaging across a large range of depth (~ 1.2 mm), such as recording population of neurons across different layers of mouse brains. The technology developed within this program will have potential impacts in a large number of biomedical fields such as neuroscience, immunology, and cancer biology.