3.4 Tilt Series Acquisition
When suitable grids have been obtained, tilt series can be collected. The ease of
collection, quality, and characteristics of the data collected depends on the hardware
configuration of the microscope and selection of parameters. In this section we step
through the implications of hardware configuration, software configuration, and
highlight the rationale behind choice of various data collection parameters.
3.4.1 Hardware Configuration
The user is likely to have a choice of microscopes to collect data on, either at a local
facility, or by traveling to collect data at a remote facility. This section provides an
overview of contemporary electron microscope hardware to enable the user to
assess the suitability of a microscope for a given project.
The first step is loading the specimen into the microscope itself. Loading into the
microscope needs to be rapid and controlled so as to avoid warming or exposure to
moisture in the atmosphere that might condense onto the specimen during transfer;
if vitreous ice warms above −160 °C [33] it may undergo a phase transition to
cubic ice. Loading and transfer of grids focuses, therefore, on maintaining the grid
either in liquid nitrogen or in the supercooled gaseous nitrogen immediately above
the liquid nitrogen surface which excludes atmospheric water.
Two main types of specimen loading options are available in commercial
microscopes: side-entry cryoholder systems enable insertion of a single grid into the
microscope at once, while cryocartridge systems allow the user to insert multiple
grids simultaneously into a holding area in the column. With cryoholder systems,
grids are mounted on the end of a cooled metal rod inserted through an airlock in
the side of the microscope, necessitating brief exposure to the surrounding atmosphere protected only by a thin retractable shutter. This is generally seen as less
stable and lower throughput than cartridge entry because the sample is in direct
physical contact with a large liquid nitrogen dewar with the possibility of vibrations
from boiling nitrogen. Nevertheless acquisition of high quality data is possible on
side entry microscopes given a well-maintained stage. With a cryocartridge system
grids are cooled by thermocoupling to the microscope cryogen via flexible braids
instead of physical attachment to the cryoholder dewar itself. With these systems,
vibrations are reduced, and data collection is considered to be more stable. After
insertion, the cryoholder’s dewar must be refilled every few hours, whereas with
cartridge-based systems the cryogen dewar is not directly attached to the specimen
holder and can therefore be considerably larger, facilitating longer data collection
sessions between cryogen refills, which can be automated. High throughput can
also be devised for cryoholder-based systems by building automated robotic
refilling strategies controlled by data collection software [34, 35]. It was thought
that using liquid helium as a microscope cryogen might provide some benefits by
68
J. L. Ferreira et al.
When suitable grids have been obtained, tilt series can be collected. The ease of
collection, quality, and characteristics of the data collected depends on the hardware
configuration of the microscope and selection of parameters. In this section we step
through the implications of hardware configuration, software configuration, and
highlight the rationale behind choice of various data collection parameters.
3.4.1 Hardware Configuration
The user is likely to have a choice of microscopes to collect data on, either at a local
facility, or by traveling to collect data at a remote facility. This section provides an
overview of contemporary electron microscope hardware to enable the user to
assess the suitability of a microscope for a given project.
The first step is loading the specimen into the microscope itself. Loading into the
microscope needs to be rapid and controlled so as to avoid warming or exposure to
moisture in the atmosphere that might condense onto the specimen during transfer;
if vitreous ice warms above −160 °C [33] it may undergo a phase transition to
cubic ice. Loading and transfer of grids focuses, therefore, on maintaining the grid
either in liquid nitrogen or in the supercooled gaseous nitrogen immediately above
the liquid nitrogen surface which excludes atmospheric water.
Two main types of specimen loading options are available in commercial
microscopes: side-entry cryoholder systems enable insertion of a single grid into the
microscope at once, while cryocartridge systems allow the user to insert multiple
grids simultaneously into a holding area in the column. With cryoholder systems,
grids are mounted on the end of a cooled metal rod inserted through an airlock in
the side of the microscope, necessitating brief exposure to the surrounding atmosphere protected only by a thin retractable shutter. This is generally seen as less
stable and lower throughput than cartridge entry because the sample is in direct
physical contact with a large liquid nitrogen dewar with the possibility of vibrations
from boiling nitrogen. Nevertheless acquisition of high quality data is possible on
side entry microscopes given a well-maintained stage. With a cryocartridge system
grids are cooled by thermocoupling to the microscope cryogen via flexible braids
instead of physical attachment to the cryoholder dewar itself. With these systems,
vibrations are reduced, and data collection is considered to be more stable. After
insertion, the cryoholder’s dewar must be refilled every few hours, whereas with
cartridge-based systems the cryogen dewar is not directly attached to the specimen
holder and can therefore be considerably larger, facilitating longer data collection
sessions between cryogen refills, which can be automated. High throughput can
also be devised for cryoholder-based systems by building automated robotic
refilling strategies controlled by data collection software [34, 35]. It was thought
that using liquid helium as a microscope cryogen might provide some benefits by
68
J. L. Ferreira et al.