Chapter 3
Practical Approaches for Cryo-FIB Milling and Applications
for Cellular Cryo-Electron Tomography
Vinson Lam and Elizabeth Villa
Abstract
Cryo-electron tomography (cryo-ET) is a powerful technique to examine cellular structures as they exist in
situ. However, direct imaging by TEM for cryo-ET is limited to specimens up to $400 nm in thickness,
narrowing its applicability to areas such as cellular projections or small bacteria and viruses. Cryo-focused
ion beam (cryo-FIB) milling has emerged in recent years as a method to generate thin specimens from
cellular samples in preparation for cryo-ET. In this technique, specimens are thinned with a beam of gallium
ions to gradually ablate cellular material in order to leave a thin, electron-transparent section (a lamella)
through the bulk material. The lamella can be used for high-resolution cryo-ET to visualize cells in 3D in a
near-native state. This approach has proved to be robust and relatively simple for new users and exhibits
minimal sectioning artifacts. In this chapter, we describe a general approach to cryo-FIB milling for users
with prior cryo-EM experience, with extensive notes on operation and troubleshooting.
Key words Cryo-focused ion beam milling, Cryo-electron tomography, Sample preparation, Lamella,
Mammalian cells, Yeast, Bacteria
1 Introduction
A major goal in biological imaging is to visualize macromolecular
complexes and the intricate networks they form at high-resolution
in situ with minimal artifacts. However, most commonly accessible
techniques lack sufficient resolution (such as fluorescence light
microscopy) or sufficient cellular context (such as single particle
cryo-EM). Cryo-electron tomography is a high-resolution imaging
modality that can visualize macromolecular structures in situ in a
near-native state in their cellular context [1]. However, specimen
thickness is a major limitation with cryo-ET and other electron
microscopy techniques. Due to multiple electron scattering, specimen thickness is limited to less than 400 nm at 300 kV [2], which is
well below the size of eukaryotic cells and most bacteria.
One approach to overcome this limitation involves sectioning
cells under cryogenic conditions using a microtome [3]. However,
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_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
49
Practical Approaches for Cryo-FIB Milling and Applications
for Cellular Cryo-Electron Tomography
Vinson Lam and Elizabeth Villa
Abstract
Cryo-electron tomography (cryo-ET) is a powerful technique to examine cellular structures as they exist in
situ. However, direct imaging by TEM for cryo-ET is limited to specimens up to $400 nm in thickness,
narrowing its applicability to areas such as cellular projections or small bacteria and viruses. Cryo-focused
ion beam (cryo-FIB) milling has emerged in recent years as a method to generate thin specimens from
cellular samples in preparation for cryo-ET. In this technique, specimens are thinned with a beam of gallium
ions to gradually ablate cellular material in order to leave a thin, electron-transparent section (a lamella)
through the bulk material. The lamella can be used for high-resolution cryo-ET to visualize cells in 3D in a
near-native state. This approach has proved to be robust and relatively simple for new users and exhibits
minimal sectioning artifacts. In this chapter, we describe a general approach to cryo-FIB milling for users
with prior cryo-EM experience, with extensive notes on operation and troubleshooting.
Key words Cryo-focused ion beam milling, Cryo-electron tomography, Sample preparation, Lamella,
Mammalian cells, Yeast, Bacteria
1 Introduction
A major goal in biological imaging is to visualize macromolecular
complexes and the intricate networks they form at high-resolution
in situ with minimal artifacts. However, most commonly accessible
techniques lack sufficient resolution (such as fluorescence light
microscopy) or sufficient cellular context (such as single particle
cryo-EM). Cryo-electron tomography is a high-resolution imaging
modality that can visualize macromolecular structures in situ in a
near-native state in their cellular context [1]. However, specimen
thickness is a major limitation with cryo-ET and other electron
microscopy techniques. Due to multiple electron scattering, specimen thickness is limited to less than 400 nm at 300 kV [2], which is
well below the size of eukaryotic cells and most bacteria.
One approach to overcome this limitation involves sectioning
cells under cryogenic conditions using a microtome [3]. However,
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_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
49