13. Care must be taken using this method for determining Zheight since the focused beam will compromise some radiation
sensitive specimens (i.e., thin ice, resins, etc.). It is recommended that procedures steps 2a or 2b (Subheading 3.1) be
subsequently used to fine-tune eucentric height.
14. Method step 2c (Subheading 3.1) for bringing the specimen to
eucentric height provides a means to quickly check basic alignments of the objective lens through evaluation of the caustic
ring.
(a) Rotation center: If rotation center is grossly misaligned,
then the center of the spot will not be located at the center
of the caustic ring. Adjust rotation center until the center
of the spot lies at the center of the caustic ring.
(b) Objective astigmatism: If the objective lens is astigmatic,
then the shape of the caustic ring will no longer be a circle.
Adjust the currents through the objective lens stigmators
until the caustic ring is round.
15. Tecnai Image Acquisition can also be used to view the live FFT
of an image acquired using the CCD camera although this
method is less sensitive than using a direct electron detector,
and higher exposure rates should be used.
16. Misalignment of beam tilt pivot points can result in autofocus
errors when using automated software packages. In practice,
most autofocus features implemented in automated data collection software packages (i.e., Leginon [14], EPU [16], SerialEM [15]) estimate focus within 200 nm or better of proper
focus.
17. The degree of objective astigmatism changes with the amount
of defocus introduced [17]. To ensure the amount of astigmatism present in the Contrast Transfer Function (CTF) is minimized during imaging of the specimen, stigmation of the
objective lens should be performed at the mean defocus value
planned for data collection. For conventional imaging using
the Talos Arctica, underfocus values between 400 and
1500 nm are recommended [11, 13].
18. In addition to manual minimization of objective astigmatism
using Digital Micrograph
® or the program s
2 stigmator [18],
automated measurement and correction of astigmatism are
implemented within Leginon [14], EPU [16], and
SerialEM [15].
19. Whenever the objective aperture is removed, inserted, or
recentered, it is best to measure and correct for any astigmatism
as the physical movement of the aperture motor is imperfect.
20. Significant changes in the rotation center alignment will alter
the centering of the objective aperture in the back focal plane
of the objective lens. Although the aperture has not physically
Parallel Illumination on the Talos Arctica
141
sensitive specimens (i.e., thin ice, resins, etc.). It is recommended that procedures steps 2a or 2b (Subheading 3.1) be
subsequently used to fine-tune eucentric height.
14. Method step 2c (Subheading 3.1) for bringing the specimen to
eucentric height provides a means to quickly check basic alignments of the objective lens through evaluation of the caustic
ring.
(a) Rotation center: If rotation center is grossly misaligned,
then the center of the spot will not be located at the center
of the caustic ring. Adjust rotation center until the center
of the spot lies at the center of the caustic ring.
(b) Objective astigmatism: If the objective lens is astigmatic,
then the shape of the caustic ring will no longer be a circle.
Adjust the currents through the objective lens stigmators
until the caustic ring is round.
15. Tecnai Image Acquisition can also be used to view the live FFT
of an image acquired using the CCD camera although this
method is less sensitive than using a direct electron detector,
and higher exposure rates should be used.
16. Misalignment of beam tilt pivot points can result in autofocus
errors when using automated software packages. In practice,
most autofocus features implemented in automated data collection software packages (i.e., Leginon [14], EPU [16], SerialEM [15]) estimate focus within 200 nm or better of proper
focus.
17. The degree of objective astigmatism changes with the amount
of defocus introduced [17]. To ensure the amount of astigmatism present in the Contrast Transfer Function (CTF) is minimized during imaging of the specimen, stigmation of the
objective lens should be performed at the mean defocus value
planned for data collection. For conventional imaging using
the Talos Arctica, underfocus values between 400 and
1500 nm are recommended [11, 13].
18. In addition to manual minimization of objective astigmatism
using Digital Micrograph
® or the program s
2 stigmator [18],
automated measurement and correction of astigmatism are
implemented within Leginon [14], EPU [16], and
SerialEM [15].
19. Whenever the objective aperture is removed, inserted, or
recentered, it is best to measure and correct for any astigmatism
as the physical movement of the aperture motor is imperfect.
20. Significant changes in the rotation center alignment will alter
the centering of the objective aperture in the back focal plane
of the objective lens. Although the aperture has not physically
Parallel Illumination on the Talos Arctica
141