Project Summary/Abstract Volume electron microscopy is the only technique to-date that provides both sufficient resolution (<20 nm) and sufficient field of view (>100 μm) for the dense reconstruction of neuronal wiring diagrams. Currently, there exist two systems that have already delivered mm3-sized synaptic resolution electron microscopy stacks: Multi-beam scanning electron microscopy(Eberle et al. 2015; Ren and Kruit 2016) (mSEM) and Gridtape-based automated transmission electron microscopy(Yin et al. 2020; Maniates-Selvin et al. 2020) (Gridtape-TEM). In mSEM, the sample is scanned with up to 91 parallel beams and an image is formed by low energy secondary electrons that are generated during scanning. Gridtape-TEM detects transmitted electrons with one or multiple fast cameras simultaneously. Both techniques currently rely on collecting and imaging thousands of ultrathin serial sections (30 - 40 nm) that are being cut with a diamond knife on an ultramicrotome. For mSEM, the sections are either collected using an automated tape collecting ultramicrotome or directly onto silicon wafers. For Gridtape-TEM, the sections are collected onto an electron-transparent film in millimeter-sized apertures on Gridtape. However, serial collection of ultrathin sections is delicate and inherently prone to failures and artifacts such as section loss, folds and cracks or knife marks. More than 50% of the errors of today’s state-of-the-art automated neuron segmentation algorithms can be attributed to missing information due to serial-sectioning. As a consequence, more than 40 hours of manual segmentation proofreading by human experts are currently required to accurately reconstruct a single cortical pyramidal cell. Some of the remaining automated segmentation issues can certainly be addressed by improving the underlying algorithms. But in order to scale dense automated neuronal circuit reconstructions to whole mouse brains with about 70 million neurons, it is necessary to significantly reduce the experimental artifacts. The collection of semi-thin sections with a thickness around 100 - 500 nm has been proposed as a much more robust alternative to ultrathin sectioning. In order to maintain or even increase the resolution in Z, these semi-thin sections could be iteratively milled and scanned in the case of mSEM or a series of images at different tilt angles could be acquired in the case of Gridtape-TEM. Here we propose to combine the commercially available multi-beam scanning transmission electron microscope FASTEM from Delmic with iterative broad ion beam milling of semi-thin sections (BIB-mSTEM). First, hundreds of semi-thin sections will be collected directly onto scintillator plates using the commercially available MagC magnetic collection system. Subsequently, these sections will be iteratively thinned and imaged by going back and forth between broad ion beam milling and imaging with FASTEM. For each section, this will produce a series of iteratively milled TEM projection images that can be used to reconstruct a high-resolution 3d stack of each section. BIB-mSTEM will be substantially more robust and reliable than mSEM and Gridtape-TEM based workflows: In contrast to Gridtape-TEM, the sections are collected onto a solid substrate and not on a fragile support film. In contrast to mSEM, BIB-mSTEM forms the image from high energy transmitted electrons that are much less sensitive to local electromagnetic fields and milling-induced irregular surface topography than low energy secondary electrons.

Project Narrative Imaging biological tissue such as brains at subcellular resolution is of fundamental importance for the understanding of biological processes. With iterative thinning multibeam transmission electron microscopy, the detailed cellular ultrastructure in very large tissue samples such as entire brains could be visualized. This will enable researchers and clinicians to visualize the biological organization and cellular interactions of entire organs at centimeter-scale down to cellular compartments at nanometer resolution.