5. The finite size of the crossover in the front focal plane of the
upper objective lens results in a spread of beam tilts at the single
point in which the beam hits the specimen. The size of the
source image, and thereby the coherence angle α can be tuned
by changing the magnification of the source. A higher gun lens
(e.g., 8 vs. 4) or a higher spot size number (e.g., 8 vs. 4) gives a
smaller source image diameter and thereby a more coherent
electron beam. The downside of these settings is the decreased
beam current.
6. The conditions for parallel illumination on a two-condenser
lens TEM result in a single C2 intensity value. This strength of
C2, in combination with spot size, C2 aperture, objective
aperture, gun lens, and magnification, determine the exposure
rate. As the C2 intensity value must remain unchanged, in
order to increase or decrease the measured exposure rate at
the camera a user must change the spot size, the size of the C2
aperture, the gun lens, and/or the magnification (see
Note 30).
7. A centered 100 μm objective aperture on a Talos Arctica
(or Glacios) allows for the visualization of the second gold
powder diffraction ring (dAu(002) ¼ 2.039 A ˚ ) and will thus
eliminate information beyond ~1.6 A ˚ resolution (Fig. 5h).
Although this setup will not limit the useable high-resolution
information available for most biological single particle cryoEM specimens, if information beyond 1.6 A ˚ resolution is
anticipated, the user should insert a larger objective aperture
(i.e., 150 μm), or remove the objective aperture entirely after
aligning the microscope (Fig. 6). However, as the objective
aperture removes high-angle inelastically scattered electrons,
imaging with a large objective aperture or no aperture at all
will decrease the observed amplitude contrast compared to
smaller objective apertures. For specimens supported by gold
foil, particular caution should be taken during imaging without
an objective aperture as considerable high-angle scattering
from the gold foil will be observed, which, can deleteriously
affect frame alignment, CTF estimation, and 3D
reconstruction.
8. The image of the source should be checked to ensure proper
gun alignment and the X-FEG is not currently undergoing a
splitting event. At a spot size of 1, 150 μm C2 aperture,
73,000 kx magnification, slightly condense the beam until a
bright spot (i.e., the source) can be observed within the central
disc of the probe. The bright spot should not touch the outer
diameter of the central disc or form a tear drop image. Highresolution imaged will not be attainable if the gun tilt/alignments are off.
Parallel Illumination on the Talos Arctica
139
upper objective lens results in a spread of beam tilts at the single
point in which the beam hits the specimen. The size of the
source image, and thereby the coherence angle α can be tuned
by changing the magnification of the source. A higher gun lens
(e.g., 8 vs. 4) or a higher spot size number (e.g., 8 vs. 4) gives a
smaller source image diameter and thereby a more coherent
electron beam. The downside of these settings is the decreased
beam current.
6. The conditions for parallel illumination on a two-condenser
lens TEM result in a single C2 intensity value. This strength of
C2, in combination with spot size, C2 aperture, objective
aperture, gun lens, and magnification, determine the exposure
rate. As the C2 intensity value must remain unchanged, in
order to increase or decrease the measured exposure rate at
the camera a user must change the spot size, the size of the C2
aperture, the gun lens, and/or the magnification (see
Note 30).
7. A centered 100 μm objective aperture on a Talos Arctica
(or Glacios) allows for the visualization of the second gold
powder diffraction ring (dAu(002) ¼ 2.039 A ˚ ) and will thus
eliminate information beyond ~1.6 A ˚ resolution (Fig. 5h).
Although this setup will not limit the useable high-resolution
information available for most biological single particle cryoEM specimens, if information beyond 1.6 A ˚ resolution is
anticipated, the user should insert a larger objective aperture
(i.e., 150 μm), or remove the objective aperture entirely after
aligning the microscope (Fig. 6). However, as the objective
aperture removes high-angle inelastically scattered electrons,
imaging with a large objective aperture or no aperture at all
will decrease the observed amplitude contrast compared to
smaller objective apertures. For specimens supported by gold
foil, particular caution should be taken during imaging without
an objective aperture as considerable high-angle scattering
from the gold foil will be observed, which, can deleteriously
affect frame alignment, CTF estimation, and 3D
reconstruction.
8. The image of the source should be checked to ensure proper
gun alignment and the X-FEG is not currently undergoing a
splitting event. At a spot size of 1, 150 μm C2 aperture,
73,000 kx magnification, slightly condense the beam until a
bright spot (i.e., the source) can be observed within the central
disc of the probe. The bright spot should not touch the outer
diameter of the central disc or form a tear drop image. Highresolution imaged will not be attainable if the gun tilt/alignments are off.
Parallel Illumination on the Talos Arctica
139