Chapter 6
Setting Up Parallel Illumination on the Talos Arctica
for High-Resolution Data Collection
Mark A. Herzik Jr
Abstract
Illuminating a specimen with a parallel electron beam is critical for many experiments in transmission
electron microscopy as deviations from this condition cause considerable deterioration of image quality.
Carefully establishing parallel illumination is particularly important on two-condenser lens transmission
electron microscopes (TEMs) as the parallel illumination condition is limited to a single beam intensity
value on these instruments. It was recently shown that a Thermo Fisher Scientific Talos Arctica, a
two-condenser lens TEM operating at 200 kV, equipped with a Gatan K2 Summit direct electron detector
is capable of resolving frozen-hydrated macromolecules of various sizes and internal symmetries to better
than 3 A ˚ resolution using single particle methodologies. A critical aspect of the success of these findings was
the careful alignment of the electron microscope to ensure the specimen was illuminated with a parallel
electron beam. Here, this chapter describes how to establish parallel illumination conditions in a Talos
Arctica TEM for high-resolution cryogenic data collection for structure determination.
Key words Single-particle electron cryomicroscopy, Parallel illumination, Two-condenser lens electron microscope, Talos Arctica
1 Introduction
For both single particle analysis electron cryomicroscopy (cryoEM) and electron cryotomography, it is of paramount importance
to illuminate the specimen using a parallel electron beam so as to
minimize deterioration in image quality [1, 2]. For modern transmission electron microscopes (TEMs), the strong pre-field of
condenser-objective lenses requires a crossover in the front focal
plane of the upper objective lens to ensure that the
non-isoplanatism β angle, the angle with which the electron beam
interacts with the specimen, is zero—this gives parallel illumination
of the specimen [3]. Conditions in which the source image lies
above the front focal plane of the objective lens (i.e., convergent
illumination) or below it (i.e., divergent illumination) result in an
electron beam with a non-zero β angle (Fig. 1) [3]. Due to the
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_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
125
Setting Up Parallel Illumination on the Talos Arctica
for High-Resolution Data Collection
Mark A. Herzik Jr
Abstract
Illuminating a specimen with a parallel electron beam is critical for many experiments in transmission
electron microscopy as deviations from this condition cause considerable deterioration of image quality.
Carefully establishing parallel illumination is particularly important on two-condenser lens transmission
electron microscopes (TEMs) as the parallel illumination condition is limited to a single beam intensity
value on these instruments. It was recently shown that a Thermo Fisher Scientific Talos Arctica, a
two-condenser lens TEM operating at 200 kV, equipped with a Gatan K2 Summit direct electron detector
is capable of resolving frozen-hydrated macromolecules of various sizes and internal symmetries to better
than 3 A ˚ resolution using single particle methodologies. A critical aspect of the success of these findings was
the careful alignment of the electron microscope to ensure the specimen was illuminated with a parallel
electron beam. Here, this chapter describes how to establish parallel illumination conditions in a Talos
Arctica TEM for high-resolution cryogenic data collection for structure determination.
Key words Single-particle electron cryomicroscopy, Parallel illumination, Two-condenser lens electron microscope, Talos Arctica
1 Introduction
For both single particle analysis electron cryomicroscopy (cryoEM) and electron cryotomography, it is of paramount importance
to illuminate the specimen using a parallel electron beam so as to
minimize deterioration in image quality [1, 2]. For modern transmission electron microscopes (TEMs), the strong pre-field of
condenser-objective lenses requires a crossover in the front focal
plane of the upper objective lens to ensure that the
non-isoplanatism β angle, the angle with which the electron beam
interacts with the specimen, is zero—this gives parallel illumination
of the specimen [3]. Conditions in which the source image lies
above the front focal plane of the objective lens (i.e., convergent
illumination) or below it (i.e., divergent illumination) result in an
electron beam with a non-zero β angle (Fig. 1) [3]. Due to the
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_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
125