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

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.The proposed technology, “GridTape”, aims to break the throughput barrier of electron microscopy data acquisition. These new tools and methods will provide the required speed and reliability for large volume tissue imaging, allowing researchers to discover rules underlying connectivity between neurons and neuronal networks in the brain. Understanding the basic principles of neuronal connectivity and their relationship to function will help reveal how the brain is altered in neurological and neurodegenerative disorders such as schizophrenia, autism, and Alzheimer’s disease and point toward network-based strategies remedy them.