this approach is technically demanding and introduces visual and
structural artifacts that arise from the sectioning process [4]. Recent
work has adapted the versatile focused ion beam (FIB) instrument
for cryo-electron microscopy applications as a method to generate
thin sections through cellular material, yielding unprecedented
insight into eukaryotic and prokaryotic cell biology [5–24]. This
technique offers significant advantages including relative ease of
use, minimal artifacts, and the ability to target specific cells for
high-resolution cryo-ET.
Focused ion beam (FIB) microscopy and milling has been used
in materials science as a method to thin samples for analysis and to
create micro/nanoscale patterns [25]. The operation uses a tightly
focused (5–10 nm) beam of ions, typically gallium, that rasters over
the sample [26], similar to the beam of electrons in conventional
scanning electron microscopy (SEM). Secondary electrons are generated by the ion beam’s interaction with the sample, which can be
detected to form images. Furthermore, the relatively large mass of
gallium ions can be used as a tool for micro/nano machining by
selectively ablating material from a specimen. In this manner, the
FIB can be used to precisely remove material above and below a
region of interest to leave a thin section—a lamella—that remains
supported by the unmilled material around it. FIB milling sample
preparation is usually performed in a “Dual-Beam” instrument
with both FIB and SEM columns for simultaneous milling and
multi-perspective imaging. These instruments are also typically
equipped with gas-injection systems that allow targeted metal
deposition or modification of milling characteristics [27]. For
cryo-FIB applications, these instruments are equipped with a
stage cooled to liquid nitrogen temperatures to maintain samples
at cryogenic temperatures and an airlock quick-loading system to
introduce cryogenic specimens into the chamber under vacuum.
This allows cold samples to be transferred into and out of the
instrument while avoiding atmospheric ice contamination and to
be milled at liquid nitrogen temperatures for several hours.
This chapter will briefly describe practical principles and procedures for cryo-FIB milling including considerations for upstream
sample preparation, downstream TEM tomography, and evaluation
of sample quality as part of a typical cryo-ET workflow (Fig. 1). A
closely related technique using cryo-FIB lift-out can be used for
much thicker samples such as tissue blocks [28–30], but will not be
discussed in this chapter. At this time of writing, commercial dualbeam instruments can be outfitted with third party cryostages/
quickloader systems such as the Quorum PP3006 cryo-stage, the
Leica EM VCT500, and the Hummingbird Scientific cryotransfer
system. In this chapter, we will describe a typical FIB milling
protocol using the Thermo Fisher Scientific (TFS) Aquilos, a
dual-beam FIB/SEM platform that includes a cryo-stage/
quickloader system. However, many of the general principles
should be applicable across instruments.
50
Vinson Lam and Elizabeth Villa
structural artifacts that arise from the sectioning process [4]. Recent
work has adapted the versatile focused ion beam (FIB) instrument
for cryo-electron microscopy applications as a method to generate
thin sections through cellular material, yielding unprecedented
insight into eukaryotic and prokaryotic cell biology [5–24]. This
technique offers significant advantages including relative ease of
use, minimal artifacts, and the ability to target specific cells for
high-resolution cryo-ET.
Focused ion beam (FIB) microscopy and milling has been used
in materials science as a method to thin samples for analysis and to
create micro/nanoscale patterns [25]. The operation uses a tightly
focused (5–10 nm) beam of ions, typically gallium, that rasters over
the sample [26], similar to the beam of electrons in conventional
scanning electron microscopy (SEM). Secondary electrons are generated by the ion beam’s interaction with the sample, which can be
detected to form images. Furthermore, the relatively large mass of
gallium ions can be used as a tool for micro/nano machining by
selectively ablating material from a specimen. In this manner, the
FIB can be used to precisely remove material above and below a
region of interest to leave a thin section—a lamella—that remains
supported by the unmilled material around it. FIB milling sample
preparation is usually performed in a “Dual-Beam” instrument
with both FIB and SEM columns for simultaneous milling and
multi-perspective imaging. These instruments are also typically
equipped with gas-injection systems that allow targeted metal
deposition or modification of milling characteristics [27]. For
cryo-FIB applications, these instruments are equipped with a
stage cooled to liquid nitrogen temperatures to maintain samples
at cryogenic temperatures and an airlock quick-loading system to
introduce cryogenic specimens into the chamber under vacuum.
This allows cold samples to be transferred into and out of the
instrument while avoiding atmospheric ice contamination and to
be milled at liquid nitrogen temperatures for several hours.
This chapter will briefly describe practical principles and procedures for cryo-FIB milling including considerations for upstream
sample preparation, downstream TEM tomography, and evaluation
of sample quality as part of a typical cryo-ET workflow (Fig. 1). A
closely related technique using cryo-FIB lift-out can be used for
much thicker samples such as tissue blocks [28–30], but will not be
discussed in this chapter. At this time of writing, commercial dualbeam instruments can be outfitted with third party cryostages/
quickloader systems such as the Quorum PP3006 cryo-stage, the
Leica EM VCT500, and the Hummingbird Scientific cryotransfer
system. In this chapter, we will describe a typical FIB milling
protocol using the Thermo Fisher Scientific (TFS) Aquilos, a
dual-beam FIB/SEM platform that includes a cryo-stage/
quickloader system. However, many of the general principles
should be applicable across instruments.
50
Vinson Lam and Elizabeth Villa