Multi-Beam Scanning Electron Microscopy for High-Throughput Imaging in Connectomics Research

Abstract

Major progress has been achieved in recent years in three-dimensional microscopy techniques. This applies to the life sciences in general, but specifically the neuroscientific field has been a main driver for developments regarding volume imaging. In particular, scanning electron microscopy offers new insights into the organization of cells and tissues by volume imaging methods, such as serial section array tomography, serial block-face imaging or focused ion beam tomography. However, most of these techniques are restricted to relatively small tissue volumes due to the limited acquisition throughput of most standard imaging techniques. Recently, a novel multi-beam scanning electron microscope technology optimized to the imaging of large sample areas has been developed. Complemented by the commercialization of automated sample preparation robots, the mapping of larger, cubic millimeter range tissue volumes at high-resolution is now within reach. This Mini Review will provide a brief overview of the various approaches to electron microscopic volume imaging, with an emphasis on serial section array tomography and multi-beam scanning electron microscopic imaging.

Topological Properties of Neuromorphic Nanowire Networks

“…Furthermore, due to the networks' self-assembled structure, it is experimentally difficult to control the topology to measure how it influences dynamics. It is also extremely difficult to use imaging-based techniques such as or electron microscopy (e.g., White et al, 1986;Eberle and Zeidler, 2018) reconstructions to unpack the structural connectivity of ASNs, as it is impossible to tell whether or not intersecting wires form a junction between them. We therefore have developed a computational model that simulates the structure experimental ASNs, based on functional, experimental validation (Kuncic et al, 2018;Diaz-Alvarez et al, 2019).…”

En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics

“…In particular, our staining methods now provide an excellent compromise between specimen contrast and accelerated FIB-SEM sectioning speed. Imaging speed may be further enhanced using higher specimen contrast to yield usable images yet more quickly, however; and in the future also possibly by using gas cluster milling (Hayworth et al, 2019) combined with SEM with multi-beam imaging (Eberle and Zeidler, 2018). Even so, many sensory inputs to both brain regions are necessarily removed when their axons are severed, and these leave behind degenerating afferent axons, which yield electron-dense profiles.…”

A petascale automated imaging pipeline for mapping neuronal circuits with high-throughput transmission electron microscopy

“…This is in contrast with serial-section approaches, using either scanning or transmission electron microscopy (TEM) 7 , 10 , 11 , 22 , 23 , which provide the opportunities for reimaging. For SEM, the highest throughput has been achieved with multi-beam instruments 29 , 30 , which were originally designed for semiconductor lithography, reverse engineering, and wafer defect inspection, but recently found great potential in neuronal circuit reconstruction. The multi-beam SEMs have achieved burst imaging rates at 0.45 Gpixel per sec in mouse brain 29 , 30 , showing a remarkable advancement in acquisition speed and capability of imaging large sample areas.…”

“…For SEM, the highest throughput has been achieved with multi-beam instruments 29 , 30 , which were originally designed for semiconductor lithography, reverse engineering, and wafer defect inspection, but recently found great potential in neuronal circuit reconstruction. The multi-beam SEMs have achieved burst imaging rates at 0.45 Gpixel per sec in mouse brain 29 , 30 , showing a remarkable advancement in acquisition speed and capability of imaging large sample areas. There are however factors that lead to long-term effective continuous acquisition rates still yet to be demonstrated, such as stage motion accuracy and settling time, focus variation across montage, system stability for continuous 24/7 operation, and maintenance overhead.…”