2 Materials
1. 500 nm crossed lines grating replica calibration grid (see
Note 1).
2. Autogrid C-clip rings (Thermo Fisher Scientific, Product
#1036173) and Autogrid C-clips (Thermo Fisher Scientific
Product #1036171) for Talos Arctica TEM autoloader.
2.1 Equipment
1. Thermo Fisher Scientific Talos Arctica (or Glacios) TEM (see
Note 2) operating at 200 keV equipped with a Gatan K2
Summit direct electron detector and a 4 K Â 4 K CETA
16 M CCD camera (or similar, see Note 3), operating with
the following microscope settings:
(a) Extraction voltage: 4150 V (see Note 4).
(b) Gun lens setting: 8 (see Notes 5 and 6).
(c) Spot size 3 (see Notes 5 and 6).
(d) C1 aperture: 2000 μm.
(e) C2 aperture: 70 μm (see Note 6).
(f) Objective aperture: 100 μm (see Note 7).
2.2 Software
1. Tecnai User Interface (Thermo Fisher Scientific).
2. Tecnai Imaging & Analysis (Thermo Fisher Scientific).
3. Gatan Microscopy Suite
® (Gatan, Inc.) which includes Digital
Micrograph
® .
4. Leginon [14], SerialEM [15], or EPU [16].
3 Methods
3.1 Basic Column
Alignments
This protocol assumes the following: (a) that basic column alignments, including gun alignments (see Note 8), pivot points, beam
and image shifts, and lens calibrations have already been performed,
(b) the Tecnai User Interface/Tecnai Imaging & Analysis and
Digital Micrograph
® software packages are installed on the scope
and camera computers, respectively, (c) a standard crossed lines
grating replica calibration grid has been clipped and loaded into
the compustage of the microscope (see Note 9), and (d) all SA
mode magnifications (e.g., SA 45,000Â, 73,000Â, or 120,000Â)
use Nano probe mode while all M and LM mode magnifications use
Micro probe mode (see Note 10).
1. Set the microscope to a lower magnification (e.g., LM-280Â)
and use the trackball to move the stage to the center of an intact
square on the calibration grid that is devoid of obvious physical
Parallel Illumination on the Talos Arctica
131
1. 500 nm crossed lines grating replica calibration grid (see
Note 1).
2. Autogrid C-clip rings (Thermo Fisher Scientific, Product
#1036173) and Autogrid C-clips (Thermo Fisher Scientific
Product #1036171) for Talos Arctica TEM autoloader.
2.1 Equipment
1. Thermo Fisher Scientific Talos Arctica (or Glacios) TEM (see
Note 2) operating at 200 keV equipped with a Gatan K2
Summit direct electron detector and a 4 K Â 4 K CETA
16 M CCD camera (or similar, see Note 3), operating with
the following microscope settings:
(a) Extraction voltage: 4150 V (see Note 4).
(b) Gun lens setting: 8 (see Notes 5 and 6).
(c) Spot size 3 (see Notes 5 and 6).
(d) C1 aperture: 2000 μm.
(e) C2 aperture: 70 μm (see Note 6).
(f) Objective aperture: 100 μm (see Note 7).
2.2 Software
1. Tecnai User Interface (Thermo Fisher Scientific).
2. Tecnai Imaging & Analysis (Thermo Fisher Scientific).
3. Gatan Microscopy Suite
® (Gatan, Inc.) which includes Digital
Micrograph
® .
4. Leginon [14], SerialEM [15], or EPU [16].
3 Methods
3.1 Basic Column
Alignments
This protocol assumes the following: (a) that basic column alignments, including gun alignments (see Note 8), pivot points, beam
and image shifts, and lens calibrations have already been performed,
(b) the Tecnai User Interface/Tecnai Imaging & Analysis and
Digital Micrograph
® software packages are installed on the scope
and camera computers, respectively, (c) a standard crossed lines
grating replica calibration grid has been clipped and loaded into
the compustage of the microscope (see Note 9), and (d) all SA
mode magnifications (e.g., SA 45,000Â, 73,000Â, or 120,000Â)
use Nano probe mode while all M and LM mode magnifications use
Micro probe mode (see Note 10).
1. Set the microscope to a lower magnification (e.g., LM-280Â)
and use the trackball to move the stage to the center of an intact
square on the calibration grid that is devoid of obvious physical
Parallel Illumination on the Talos Arctica
131