C2–C3 lenses (i.e., beam intensity) (Fig. 4). Although
two-condenser lens TEMs, such as the Thermo Fisher Scientific
Talos Arctica, utilize a C1–C2 condenser zoom system, the absence
of a third condenser lens means that the parallel illumination condition is only guaranteed for a single beam intensity value as the
strength of the C2 lens controls the height of the source image in
front of the upper objective lens. As a result, when collecting data
using a two-condenser lens TEM, it is imperative that the C2 lens
strength is properly set to confer parallel illumination (Fig. 4).
Critical to determining the parallel illumination condition in a
TEM is aligning the source image after the last condenser lens (i.e.,
C2 for a two-condenser lens TEM) with the front focal plane of the
upper objective lens (Fig. 4) [10]. Although the front focal plane
cannot be viewed directly, the symmetrical nature of modern split
objective lenses, wherein the specimen lies between the upper and
lower pole pieces of the objective lens, means that the height of the
front focal plane from the upper pole piece is approximately the
same distance as the back focal plane from the lower pole piece of
the objective lens. As a result, the procedure for determining parallel illumination relies on maximizing the “sharpness” of diffraction
spots when the back focal plane of the lower objective lens is
projected onto the viewing plane. To accomplish this, the objective
aperture, which lies in the back focal plane of the objective lens,
must first be brought into focus in diffraction mode. By adjusting
beam intensity to maximize the sharpness of gold powder diffraction rings, such as those obtained from a crossed lines grating
replica calibration grid containing sputtered gold, the source
image will be aligned to the front focal plane. Importantly, for
two-condenser lens TEMs, this singular beam intensity value
must be used for data collection; otherwise, image quality will
suffer from the effects of convergent or divergent illumination.
It was recently shown that the Thermo Fisher Scientific Talos
Arctica TEM operating at 200 kV coupled with a Gatan K2 Summit
direct electron detector is capable of resolving frozen-hydrated
macromolecules to better than 3 A ˚ resolution using single particle
methodologies [11–13]. Critical to this success was the careful
alignment of the electron microscope to ensure ideal illumination
of the specimen using a parallel electron beam [12]. This protocol
details how to establish parallel illumination conditions for a
two-condenser lens TEM, such as the Talos Arctica, in preparation
for high-resolution data collection using a K2 Summit direct electron detector. This protocol also includes instructions for determining the optimal exposure rate of the specimen while ensuring
the parallel illumination condition is maintained.
130
Mark A. Herzik Jr
two-condenser lens TEMs, such as the Thermo Fisher Scientific
Talos Arctica, utilize a C1–C2 condenser zoom system, the absence
of a third condenser lens means that the parallel illumination condition is only guaranteed for a single beam intensity value as the
strength of the C2 lens controls the height of the source image in
front of the upper objective lens. As a result, when collecting data
using a two-condenser lens TEM, it is imperative that the C2 lens
strength is properly set to confer parallel illumination (Fig. 4).
Critical to determining the parallel illumination condition in a
TEM is aligning the source image after the last condenser lens (i.e.,
C2 for a two-condenser lens TEM) with the front focal plane of the
upper objective lens (Fig. 4) [10]. Although the front focal plane
cannot be viewed directly, the symmetrical nature of modern split
objective lenses, wherein the specimen lies between the upper and
lower pole pieces of the objective lens, means that the height of the
front focal plane from the upper pole piece is approximately the
same distance as the back focal plane from the lower pole piece of
the objective lens. As a result, the procedure for determining parallel illumination relies on maximizing the “sharpness” of diffraction
spots when the back focal plane of the lower objective lens is
projected onto the viewing plane. To accomplish this, the objective
aperture, which lies in the back focal plane of the objective lens,
must first be brought into focus in diffraction mode. By adjusting
beam intensity to maximize the sharpness of gold powder diffraction rings, such as those obtained from a crossed lines grating
replica calibration grid containing sputtered gold, the source
image will be aligned to the front focal plane. Importantly, for
two-condenser lens TEMs, this singular beam intensity value
must be used for data collection; otherwise, image quality will
suffer from the effects of convergent or divergent illumination.
It was recently shown that the Thermo Fisher Scientific Talos
Arctica TEM operating at 200 kV coupled with a Gatan K2 Summit
direct electron detector is capable of resolving frozen-hydrated
macromolecules to better than 3 A ˚ resolution using single particle
methodologies [11–13]. Critical to this success was the careful
alignment of the electron microscope to ensure ideal illumination
of the specimen using a parallel electron beam [12]. This protocol
details how to establish parallel illumination conditions for a
two-condenser lens TEM, such as the Talos Arctica, in preparation
for high-resolution data collection using a K2 Summit direct electron detector. This protocol also includes instructions for determining the optimal exposure rate of the specimen while ensuring
the parallel illumination condition is maintained.
130
Mark A. Herzik Jr