"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 neuroscienceExtracting a circuit sized volume at synaptic resolution requires large scalehigh throughput imaging of many thin sections of brain tissueThe immediate goal of researchers is to capture and map a cubic millimeter of cortex at synaptic resolutionrequiring the collection and imaging oftissue sections for a single datasetThe slow speed of tissue sectioning and imaging remains a major limitation to the field of connectomicsrequiring many years to acquire a dataset of this sizeA number of research intuitionse gHarvard UniversityJanelia Research CampusAllen Instituteare actively seeking solutions for higherthroughput imagingScanning electron microscopySEMoffers fastreliable sample sectioning and collection via Automatic Tape collecting Ultra MicrotomeATUM SEMas well as automated in vacuum sample cutting via Serial Block FaceSBF SEMor Focused Ion BeamFIB SEMHoweverSEM has intrinsically poor resolution and signal leveldue to the serial nature of the scanning electron beam pixel collectionIn comparisoncamera based transmission electron microscopyTEMallows fastsimultaneous collection of millions of pixels at higher resolution than SEMbut is limited by slow handling of individual sample supportsTo overcome this throughput limitationresearchers at Harvard University are developing a tape sample substrate for TEM calledGridTapecomprising thin film covered slots in a tape form that is compatible with commercially available ATUM equipmentSections are imaged via an in column reel to reel TEM imaging stageHarvard has chosen Luxel as their partner for the initial investigation and demonstratedX speed improvement in pickup of thin sections of mouse and Drosophila melanogaster brains cut with an ATUMThe proposed project will optimize and scale up GridTape to meet the urgent unmet needs of large scale connectomics researchThe technical challenges include reel to reel thin film laminationfilm breakagewrinklingand image background noiseThe specific aims of this proposal areDevelop a pilot manufacturing process for the production of GridTape prototypes having the number of imaging slots required by researchersin a continuous lengthwith production throughput ofslots dayOptimize GridTape specifications to meet end user requirementsreducing damaged slot rate to ltandConnectomics field evaluationacquiring datasets on GridTape prototypes have rtslots and a quantitative comparison between GridTape and SEM datasets demonstrating pickup speed and imaging throughput at least equal to existing practicePhase II will address manufacturing scale up with greater slot counts and lower cost per slot