Chapter 2
Cryo-Electron Tomography and Automatic Segmentation
of Cultured Hippocampal Neurons
Ryan K. Hylton, Victoria H. Seader, and Matthew T. Swulius
Abstract
Cryo-electron tomography is fast becoming a preferred method for studying intracellular environments at
the molecular scale. Increases in data collection throughput means that large numbers of tomograms can be
generated at rates too fast for humans to easily explore quantitatively. Currently, there is a large effort to
make data collection and segmentation tools more automated. Here, we describe a workflow for preparing
cultured neurons on electron microscopy grids, batch tomographic data collection, reconstruction and
automatic segmentation using freely and commercially available software.
Key words Cryo, Cryo-EM, Cryo-ET, ECT, CET, Tomography, Neuron, Segmentation, Neural
Network, IMOD, EMAN2
1 Introduction
While all cells are fascinating, the neuron is of specific importance to
the diversity of animal behavior and psychological experience. It is
not surprising, then, that they take on very complex morphologies
themselves and have much to reveal about the structural mechanisms underlying many dynamic cellular processes. Fortunately,
cryo-electron tomography (cryo-ET) is a useful method for imaging the fine biological architecture of neurons, where the direct
imaging of cryo-preserved cytoplasm allows for unprecedented
three-dimensional clarity at the molecular scale [1, 2].
One of cryo-ETs major limitations is sample thickness. There is
a practical cut-off at approximately 1 μm, but to achieve the highest
contrast and resolution, the sample should be no more than a few
100 nm thick. Due to this, mammalian cells are typically inaccessible without the use of cryo-sectioning or cryo-focused ion milling
[3, 4]. Luckily, the long branches of neurons grown in culture are
ideal for cryo-ET (<500 nm-thick) and provide very high quality
cryotomograms reliably.
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
25
Cryo-Electron Tomography and Automatic Segmentation
of Cultured Hippocampal Neurons
Ryan K. Hylton, Victoria H. Seader, and Matthew T. Swulius
Abstract
Cryo-electron tomography is fast becoming a preferred method for studying intracellular environments at
the molecular scale. Increases in data collection throughput means that large numbers of tomograms can be
generated at rates too fast for humans to easily explore quantitatively. Currently, there is a large effort to
make data collection and segmentation tools more automated. Here, we describe a workflow for preparing
cultured neurons on electron microscopy grids, batch tomographic data collection, reconstruction and
automatic segmentation using freely and commercially available software.
Key words Cryo, Cryo-EM, Cryo-ET, ECT, CET, Tomography, Neuron, Segmentation, Neural
Network, IMOD, EMAN2
1 Introduction
While all cells are fascinating, the neuron is of specific importance to
the diversity of animal behavior and psychological experience. It is
not surprising, then, that they take on very complex morphologies
themselves and have much to reveal about the structural mechanisms underlying many dynamic cellular processes. Fortunately,
cryo-electron tomography (cryo-ET) is a useful method for imaging the fine biological architecture of neurons, where the direct
imaging of cryo-preserved cytoplasm allows for unprecedented
three-dimensional clarity at the molecular scale [1, 2].
One of cryo-ETs major limitations is sample thickness. There is
a practical cut-off at approximately 1 μm, but to achieve the highest
contrast and resolution, the sample should be no more than a few
100 nm thick. Due to this, mammalian cells are typically inaccessible without the use of cryo-sectioning or cryo-focused ion milling
[3, 4]. Luckily, the long branches of neurons grown in culture are
ideal for cryo-ET (<500 nm-thick) and provide very high quality
cryotomograms reliably.
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
25