Large-scale neuroanatomy using LASSO: Loop-based Automated Serial Sectioning Operation

Abstract

Serial section transmission electron microscopy (ssTEM) is the most promising tool for investigating the three-dimensional anatomy of the brain with nanometer resolution. Yet as the field progresses to larger volumes of brain tissue, new methods for high-yield, low-cost, and high-throughput serial sectioning are required. Here, we introduce LASSO (Loop-based Automated Serial Sectioning Operation), in which serial sections are processed in “batches.” Batches are quantized groups of individual sections that, in LASSO, are cut with a diamond knife, picked up from an attached waterboat, and placed onto microfabricated TEM substrates using rapid, accurate, and repeatable robotic tools. Additionally, we introduce mathematical models for ssTEM with respect to yield, throughput, and cost to access ssTEM scalability. To validate the method experimentally, we processed 729 serial sections of human brain tissue (~40 nm x 1 mm x 1 mm). Section yield was 727/729 (99.7%). Sections were placed accurately and repeatably (x-direction: -20 ± 110 μm (1 s.d.), y-direction: 60 ± 150 μm (1 s.d.)) with a mean cycle time of 43 s ± 12 s (1 s.d.). High-magnification (2.5 nm/px) TEM imaging was conducted to measure the image quality. We report no significant distortion, information loss, or substrate-derived artifacts in the TEM images. Quantitatively, the edge spread function across vesicle edges and image contrast were comparable, suggesting that LASSO does not negatively affect image quality. In total, LASSO compares favorably with traditional serial sectioning methods with respect to throughput, yield, and cost for large-scale experiments, and represents a flexible, scalable, and accessible technology platform to enable the next generation of neuroanatomical studies.

Reconstruction of motor control circuits in adultDrosophilausing automated transmission electron microscopy

“…Compared to SEM, transmission EM (TEM) allows for higher spatial resolution (Merk et al, 2016), an order of magnitude greater signal-to-noise at the same electron dose (Xu et al, 2017;Zheng et al, 2018), and straightforward parallelization (Bock et al, 2011;Lee et al, 2016;Tobin et al, 2017;Zheng et al, 2018). Although there have been recent developments in motorized TEM section collection (Lee et al, 2018) and automated high-throughput TEM imaging (Zheng et al, 2018), we lack an end-to-end platform for automated high-throughput TEM section collection and imaging. To address this, we designed a tapebased data acquisition pipeline that combines automated sectioning with a novel TEM-compatible collection substrate and an automated, reel-to-reel imaging stage.…”

Capillary-Based and Stokes-Based Trapping of Serial Sections for Scalable 3D-EM Connectomics

“…3C,D, blue triangles) experiments shows good alignment our mathematical model without any fitted parameters (RMSE = 0.27 mm, RMSE = 0.31 mm, trap height and width studies, respectively), indicating that the sections are predominantly trapped via a balance of curvature-induced capillary interactions and Stokes-based hydrodynamic forces. From prior literature, it is likely that the curvatureinduced capillary interactions are quadrupolar-monopolar in nature (Stamou et al, 2000;Cavallaro et al, 2011;Yao et al, 2015;Lee et al, 2018a). We note that while a trap height of 0.25 mm was tested, this trap height failed to consistently trap sections.…”

“…In the following, we describe an automated, mesoscale serial sectioning platform that uses a curvature-induced capillary-interaction-based (i.e., capillary-based), and hydrodynamic force-based (i.e., Stokes-based) trap to passively constrain sections with high accuracy and repeatability. In this method, individual sections are constrained in a stable force-equilibrium state akin to that of kinematic couplings used in precision machine design (Slocum, 1992;Rothenhöfer et al, 2013;Lee et al, 2018a). Subsequently, the sections are picked-up with a loop end-effector that is rigidly affixed to a robotic, three-axis precision linear stage system.…”

MagC, magnetic collection of ultrathin sections for volumetric correlative light and electron microscopy

“…These unwanted effects typically arise when the water surface shows strong convexity profiles produced by hydrophobic substrate portions and walls, as sketched in Figure 2—figure supplement 5f. The three main precepts to avoid folding or warping drawn from my own experience and from the literature (Kubota et al, 2018; Horstmann et al, 2012; Burel et al, 2018; Koike et al, 2017; Templier and Hahnloser, 2019; Harris et al, 2006; Wacker et al, 2016; Lee et al, 2018) are to handle sections far from walls, to have a low contact angle between the water surface and the substrate, and to heat the substrate. These three precepts were followed in MagC by maintaining the sections in the center of the knife boat, by making the silicon wafer hydrophilic with a plasma treatment, and by heating the wafer with heating pads placed below the knife boat.…”