Capillary- and Stokes-based trapping of serial sections for scalable 3D-EM connectomics

Capillary- and Stokes-based trapping of serial sections for scalable 3D-EM connectomics, AbstractSerial section electron microscopy (ssEM), a technique where volumes of tissue can be anatomically reconstructed by imaging consecutive tissue slices, has proven to be a powerful tool for the investigation of brain anatomy., Abstract

Serial section electron microscopy (ssEM), a technique where volumes of tissue can be anatomically reconstructed by imaging consecutive tissue slices, has proven to be a powerful tool for the investigation of brain anatomy. Between the process of cutting the slices—or "sections"—and imaging them, however, handling 10°-10⁶ delicate sections remains a bottleneck in ssEM, especially for batches in the "mesoscale" regime, i.e.,10²-10³ sections. We present a tissue section handling device that transports and positions sections—accurately and repeatability—for automated, robotic section pick-up and placement onto an imaging substrate. The device interfaces with a conventional ultramicrotomy diamond knife, accomplishing in-line, exact-constraint trapping of sections with 100 µm repeatability. An associated mathematical model includes capillary- and Stokes-based forces, accurately describing observed behavior and fundamentally extends the modeling of water-air interface forces. Using the device, we demonstrate and describe the limits of reliable handling of hundreds of slices onto a variety of electron and light microscopy substrates without significant defects (n=8 datasets composed of 126 serial sections in an automated fashion with an average loss rate and throughput of 0.50% and 63 seconds/section, respectively. In total, this work represents an automated mesoscale serial sectioning system for scalable 3D-EM connectomics.

Significance Statement Serial section electron microscopy (ssEM), a technique where volumes of tissue can be anatomically reconstructed by imaging consecutive tissue slices, has proven to be a powerful tool for studying neuroanatomy. However, between the process of cutting the slices and imaging them, handling 10°-10⁶ delicate slices—or "sections"—remains a bottleneck in ssEM, especially for batches in the "mesoscale" regime, i.e.,10²-10³ sections. Here, we present a section handling device that transports and positions sections for automated, robotic section pick-up and placement onto an imaging substrate. As a part of this device, we characterize a trapping technique that utilizes curvature-induced capillary-based forces and hydrodynamic Stokes drag-based forces. In total, this work represents an automated mesoscale serial sectioning system for scalable 3D-EM connectomics.

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