Chapter 1
Getting Started with In Situ Cryo-Electron Tomography
Daniel Serwas and Karen M. Davies
Abstract
Cryo-electron tomography (cryo-ET) is an extremely powerful tool which is used to image cellular features
in their close-to-native environment at a resolution where both protein structure and membrane morphology can be revealed. Compared to conventional electron microscopy methods for biology, cryo-ET does
not include the use of potentially artifact generating agents for sample fixation or visualization. Despite its
obvious advantages, cryo-ET has not been widely adopted by cell biologists. This might originate from the
overwhelming and constantly growing number of complex ways to record and process data as well as the
numerous methods available for sample preparation. In this chapter, we will take one step back and guide
the reader through the essential steps of sample preparation using mammalian cells, as well as the basic steps
involved in data recording and processing. The described protocol will allow the reader to obtain data that
can be used for morphological analysis and precise measurements of biological structures in their cellular
environment. Furthermore, this data can be used for more elaborate structural analysis by applying further
image processing steps like subtomogram averaging, which is required to determine the structure of
proteins.
Key words Cell biology, Cellular morphology, Cryo-electron tomography, Mammalian cells, Segmentation, Structural cell biology, Tilt series, Vitrification
1 Introduction
Electron microscopy (EM) was a key driver in the establishment of
the cell biology field in the mid twentieth century as it provided
(and still provides) novel ultrastructural insights into cellular organization [1–3]. However, over the years, EM lost its popularity
potentially due to its labor-intensive sample preparation steps,
including fixation, dehydration, and heavy metal treatments,
which are prone to artifact generation [4–7]. The development of
cryo-EM has overcome these limitations as the sample preparation
process allows for imaging of rapidly frozen (vitrified) but still
hydrated biological specimens, e.g. mammalian cells, in close to
native conditions [8–12]. These samples can be imaged by cryoelectron tomography (cryo-ET), which is a specialized EM technique that can generate 3D volumes of unique cellular samples
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_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
Getting Started with In Situ Cryo-Electron Tomography
Daniel Serwas and Karen M. Davies
Abstract
Cryo-electron tomography (cryo-ET) is an extremely powerful tool which is used to image cellular features
in their close-to-native environment at a resolution where both protein structure and membrane morphology can be revealed. Compared to conventional electron microscopy methods for biology, cryo-ET does
not include the use of potentially artifact generating agents for sample fixation or visualization. Despite its
obvious advantages, cryo-ET has not been widely adopted by cell biologists. This might originate from the
overwhelming and constantly growing number of complex ways to record and process data as well as the
numerous methods available for sample preparation. In this chapter, we will take one step back and guide
the reader through the essential steps of sample preparation using mammalian cells, as well as the basic steps
involved in data recording and processing. The described protocol will allow the reader to obtain data that
can be used for morphological analysis and precise measurements of biological structures in their cellular
environment. Furthermore, this data can be used for more elaborate structural analysis by applying further
image processing steps like subtomogram averaging, which is required to determine the structure of
proteins.
Key words Cell biology, Cellular morphology, Cryo-electron tomography, Mammalian cells, Segmentation, Structural cell biology, Tilt series, Vitrification
1 Introduction
Electron microscopy (EM) was a key driver in the establishment of
the cell biology field in the mid twentieth century as it provided
(and still provides) novel ultrastructural insights into cellular organization [1–3]. However, over the years, EM lost its popularity
potentially due to its labor-intensive sample preparation steps,
including fixation, dehydration, and heavy metal treatments,
which are prone to artifact generation [4–7]. The development of
cryo-EM has overcome these limitations as the sample preparation
process allows for imaging of rapidly frozen (vitrified) but still
hydrated biological specimens, e.g. mammalian cells, in close to
native conditions [8–12]. These samples can be imaged by cryoelectron tomography (cryo-ET), which is a specialized EM technique that can generate 3D volumes of unique cellular samples
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_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3