"Grid-Tape": A High-Throughput Platform for Brain Connectomics and Nanoscale Structural Analysis

脑电路图是现代医学的基础性和基础性问题之一。 神经科学。由于这些电路的连接性对它们的功能有重要影响，因此请理解 这些网络的结构将对理解它们在健康和疾病中的作用产生重大影响。这个 主要的挑战是提取电路大小的突触分辨率的体积，这需要大规模的电子 数以千计的脑组织切片的显微成像。到目前为止，组织切片的速度很慢， 成像一直是连接学领域的一个主要限制，需要多年才能获得 这个尺码。在第一阶段开发的GridTape™通过允许快速自动成像来消除这一瓶颈 在透射电子显微镜(TEM)内的连续磁带上的数千个切片。这个新的卷轴- TO-卷轴样品基板利用基于相机的瞬变电磁的固有速度和分辨率优势来提供 与扫描电子显微镜(SEM)替代产品相比，数据采集速率更高，成本更低。在第一阶段， GridTape™成功收购立方毫米，实现了连接学的长期目标 4纳米分辨率的体积，产生超过2 PB的数据集。数据涵盖了26,500个系列 片段被分成7卷GridTape™，并用5台电子显微镜进行成像 在500M像素/S的有效成像率下平行六个月。而最佳的多光束扫描电子显微镜替代 使用GridTape™可以达到单个透射电子显微镜的3倍成像速率，成本大约高出20倍(400-600万美元) 目前世界上只有几个多波束扫描电子显微镜；这推动了批量经济-EM在很大程度上 支持将多个更便宜的TEM与GridTape™并行使用的方法。具有明确的可行性 演示了，Luxel将在第二阶段与哈佛大学合作，以改善以下几个数据质量问题 栅带™。技术上的挑战包括降低来自内部结构的背景图像噪声 支撑膜，降低了薄膜涂层的成本，减轻了由组织裂缝引起的图像伪影 和折叠，并扩大覆盖薄膜的狭缝区域，以允许更大的组织样本而不会损坏。我们计划 通过开发替代的低噪声聚合物薄膜来实现这些目标，这些薄膜也提供制造 可伸缩性优势，包括更快的基板移除。我们将与连接学研究人员在 哈佛大学和艾伦研究所确定最佳组织块制备配方和方法，以保持 减轻组织裂开和折叠的薄膜张力。

The wiring diagram of brain circuits is one of the foundational and fundamental questions of modern neuroscience. Since the connectivity of these circuits critically influences their function, understanding the structure of these networks will have a major impact on understanding their role in health and disease. The primary challenge is extracting a circuit-sized volume at synaptic resolution, which requires large-scale electron microscopy imaging of thousands of sections of brain tissue. Until now, the slow speed of tissue sectioning and imaging has been a major limitation to the field of connectomics, requiring many years to acquire a dataset of this size. GridTape™, developed in Phase I, removes this bottleneck by allowing fast automated imaging of thousands of sections on a continuous tape inside a transmission electron microscope (TEM). This new reel- to-reel sample substrate leverages inherent speed and resolution advantages of camera-based TEM to provide higher data acquisition rates with lower cost than scanning electron microscope (SEM) alternatives. In Phase I, GridTape™ achieved a longstanding goal for connectomics with the successful acquisition of a cubic mm volume at 4 nm resolution, yielding a dataset of more than 2 petabytes. The data spanned 26,500 serial sections split between seven reels of GridTape™, and was imaged with five TEM microscopes running in parallel for six months at an effective imaging rate of 500 Mpixel/s. While the best multi-beam SEM alternative can achieve 3X the imaging rate of a single TEM with GridTape™, the cost is roughly 20X higher ($4-6M) and only a few multi-beam SEMs currently exist in the world; this drives the economics of volume-EM heavily in favor of an approach using multiple cheaper TEMs in parallel with GridTape™. With feasibility clearly demonstrated, Luxel will partner with Harvard in Phase II to improve a number of data quality issues for GridTape™. The technical challenges include reducing the background image noise from intrinsic structure in the support films, reducing costs for the thin film coating, mitigating image artifacts that arise from tissue cracks and folds, and expanding the film-covered slot areas to allow larger tissue samples without breakage. We plan to achieve these goals by developing alternative low-noise polymer films that also offer manufacturing scalability benefits including faster substrate removal. We will partner with connectomics researchers at Harvard and the Allen Institute to determine optimal tissue block preparation formulas and methods to maintain film tension that mitigate tissue cracks and folds.