The wiring diagram of brain circuits is one of the foundational and fundamental questions of modern neuroscience. Extracting a circuit-sized volume at synaptic resolution requires large-scale, high-throughput imaging of many thin sections of brain tissue. The immediate goal of researchers is to capture and map a cubic millimeter of cortex at synaptic resolution, requiring the collection and imaging of 25,000 tissue sections for a single dataset. The slow speed of tissue sectioning and imaging remains a major limitation to the field of connectomics, requiring many years to acquire a dataset of this size. A number of research intuitions (e.g. Harvard University, Janelia Research Campus, Allen Institute) are actively seeking solutions for higher- throughput imaging. Scanning electron microscopy (SEM) offers fast, reliable sample sectioning and collection via Automatic Tape-collecting Ultra-Microtome (ATUM-SEM), as well as automated in-vacuum sample cutting via Serial-Block-Face (SBF-SEM) or Focused Ion Beam (FIB-SEM). However, SEM has intrinsically poor resolution and signal level, due to the serial nature of the scanning electron beam pixel collection. In comparison, camera-based transmission electron microscopy (TEM) allows fast, simultaneous collection of millions of pixels at higher resolution than SEM, but is limited by slow handling of individual sample supports. To overcome this throughput limitation, researchers at Harvard University are developing a tape sample substrate for TEM called ?GridTape?, comprising thin-film-covered slots in a tape form that is compatible with commercially available ATUM equipment. Sections are imaged via an in-column reel-to-reel TEM imaging stage. Harvard has chosen Luxel as their partner for the initial investigation and demonstrated ~70X speed improvement in pickup of thin sections of mouse and Drosophila melanogaster brains cut with an ATUM. The proposed project will optimize and scale up GridTape to meet the urgent unmet needs of large scale connectomics research. The technical challenges include reel-to-reel thin film lamination, film breakage, wrinkling, and image background noise.
The specific aims of this proposal are: (1) Develop a pilot manufacturing process for the production of GridTape prototypes having the number of imaging slots required by researchers (5,000-16,000) in a continuous length, with production throughput of 2,000 slots/day; (2) Optimize GridTape specifications to meet end-user requirements, reducing damaged slot rate to <1%; and (3) Connectomics field evaluation, acquiring datasets on GridTape prototypes have >10,000 slots and a quantitative comparison between GridTape and SEM datasets demonstrating pickup speed and imaging throughput at least equal to existing practice. Phase II will address manufacturing scale-up with greater slot counts and lower cost per slot.
The proposed technology, ?Grid Tape?, 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.
