[30], direct electron detection cameras with high quantum efficiency and the development of fast-tilt-series schemes [31] (which
decrease the time of data collection significantly) have further
extended the reach, power, and resolution of cryo-ET.
In this chapter, we present a general outline of the workflow
that we use in our laboratory to investigate different kinds of
biological samples with cryo-ET, starting from sample preparation
and proceeding through data collection and processing. This discussion will include prokaryotic as well as different types of eukaryotic samples (like plant tissues), hybrid approaches like cryo-ET
with cryo-CLEM or FIB-milling or both, and cryosectioning of
thick biological samples like plant tissues. Finally, we will comment
on the requirements for high-resolution tomography in terms of
data processing and data collection, including rapid tilt-series
schemes.
This chapter is organized into two major sections which are
sample preparation and data collection and processing. In the
sample preparation section, we will discuss bacterial samples as an
example of single small cells, isolated eukaryotic cells and plant
tissues in separate subsections. Finally, one should bear in mind
that the steps outlined here represent a general protocol and specific tweaks and modifications might be applied to each sample
specifically.
2 Sample Preparation
2.1 Single
Small Cells
This section is applicable to many types of unicellular small organisms. We will discuss bacterial samples as an example. For a detailed
discussion of how to perform tomography on a specific large bacterial protein complex, we refer the reader to our recently published
chapter [32].
2.1.1 Bacterial Cultures
In general, we start from a À80
C glycerol stock of the bacterial
strain which is cultured on a suitable agar plate under the required
conditions (e.g., temperature, antibiotics, and CO 2 ). Subsequently,
a single colony is inoculated into suitable liquid medium and
allowed to grow to the desired OD 600 . Note that this general
workflow can differ for different conditions/strains. For example,
it might be sometimes better to grow the cells on an agar plate then
resuspend them in a liquid medium just prior to freezing. Therefore, one needs to optimize the growth conditions for the investigated bacterial species and for the specific aim of the experiment.
2.1.2 Grids and Gold
Fiducials
For many bacterial samples destined for tomography, we use copper
R2/2 200 Quantifoil holey extra thick carbon grids. However, if
the cells need to be incubated on the grids for a significant amount
of time, we recommend gold grids with Quantifoil holey carbon as
copper is toxic to the cells. When correlated work between different
Methods in Cryo-Electron Tomography
85
decrease the time of data collection significantly) have further
extended the reach, power, and resolution of cryo-ET.
In this chapter, we present a general outline of the workflow
that we use in our laboratory to investigate different kinds of
biological samples with cryo-ET, starting from sample preparation
and proceeding through data collection and processing. This discussion will include prokaryotic as well as different types of eukaryotic samples (like plant tissues), hybrid approaches like cryo-ET
with cryo-CLEM or FIB-milling or both, and cryosectioning of
thick biological samples like plant tissues. Finally, we will comment
on the requirements for high-resolution tomography in terms of
data processing and data collection, including rapid tilt-series
schemes.
This chapter is organized into two major sections which are
sample preparation and data collection and processing. In the
sample preparation section, we will discuss bacterial samples as an
example of single small cells, isolated eukaryotic cells and plant
tissues in separate subsections. Finally, one should bear in mind
that the steps outlined here represent a general protocol and specific tweaks and modifications might be applied to each sample
specifically.
2 Sample Preparation
2.1 Single
Small Cells
This section is applicable to many types of unicellular small organisms. We will discuss bacterial samples as an example. For a detailed
discussion of how to perform tomography on a specific large bacterial protein complex, we refer the reader to our recently published
chapter [32].
2.1.1 Bacterial Cultures
In general, we start from a À80
C glycerol stock of the bacterial
strain which is cultured on a suitable agar plate under the required
conditions (e.g., temperature, antibiotics, and CO 2 ). Subsequently,
a single colony is inoculated into suitable liquid medium and
allowed to grow to the desired OD 600 . Note that this general
workflow can differ for different conditions/strains. For example,
it might be sometimes better to grow the cells on an agar plate then
resuspend them in a liquid medium just prior to freezing. Therefore, one needs to optimize the growth conditions for the investigated bacterial species and for the specific aim of the experiment.
2.1.2 Grids and Gold
Fiducials
For many bacterial samples destined for tomography, we use copper
R2/2 200 Quantifoil holey extra thick carbon grids. However, if
the cells need to be incubated on the grids for a significant amount
of time, we recommend gold grids with Quantifoil holey carbon as
copper is toxic to the cells. When correlated work between different
Methods in Cryo-Electron Tomography
85