2.2 Electron Detector
The electron detector, which serves as the camera for image recording purposes, should have the ability to detect and record images
with high dynamic range for accurate representation of the crystallographic diffraction patterns. It should also have a high acquisition
frame rate in order to capture as much data through the rotation
data collection as possible to minimize angular gaps in the rotation.
For this method, cameras must be compatible with SerialEM,
such as charge-coupled diode (CCD) and complementary metaloxide semiconductor (CMOS) models from Gatan, TVIPS,
Thermo Fisher, AMT, Direct Electron, and the JEOL Ruby camera. Cameras that have been tested to collect data for MicroED
using SerialEM include the TVIPS TemCam-F416, TVIPS
TemCam-XF416, Thermo Fisher Ceta (16M, 2, and D models),
Thermo Fisher Falcon III, and the Direct Electron DE-20. The
Falcon III and DE-20 are direct electron detectors; for these cases,
a beamstop was used to mask the incident beam at all times while in
diffraction mode to protect the sensor.
2.3 Image
Acquisition Software
In general, image acquisition software for the collection of
continuous-rotation MicroED data should be compatible with
both TEM and camera in order to script concerted actions for
automation. SerialEM natively supports diffraction mode and is
compatible with a wide range of microscopes and cameras. It also
includes a Low Dose Mode, which enables the user to customize
the camera settings of a set microscope lens configuration for a
given task. In this case, we will use different modes to set up (a) a
low-magnification crystal search by whole-grid atlas, (b) a Record
beam for data collection, and (c) a low-dose diffraction-defocus
beam (offset from the Record beam) [6, 7] for manually centering
the crystal as necessary.
3 Methods
3.1 Microscope
and Software Setup
A well-aligned TEM electron-optical system for imaging in the SA
magnification range is the ideal starting point for microscope alignment for the diffraction experiment [5, 8, 9]. Once switched to the
diffraction mode, the user selects the appropriate camera length,
adjusts dose (C2 intensity), and focuses the direct beam to a sharp,
condensed point via diffraction focus. Astigmatism of the diffraction beam should be checked at this point. The result is the diffraction beam used for data collection and is ultimately saved in the
SerialEM’s Low Dose Mode as the Record beam.
1. Start SerialEM. Make sure the phosphor screen is down and the
beam is visible.
2. Enable the Low Dose Mode by clicking its box under “Low
Dose Control”.
Automation of Continuous-Rotation Data Collection for MicroED
323
The electron detector, which serves as the camera for image recording purposes, should have the ability to detect and record images
with high dynamic range for accurate representation of the crystallographic diffraction patterns. It should also have a high acquisition
frame rate in order to capture as much data through the rotation
data collection as possible to minimize angular gaps in the rotation.
For this method, cameras must be compatible with SerialEM,
such as charge-coupled diode (CCD) and complementary metaloxide semiconductor (CMOS) models from Gatan, TVIPS,
Thermo Fisher, AMT, Direct Electron, and the JEOL Ruby camera. Cameras that have been tested to collect data for MicroED
using SerialEM include the TVIPS TemCam-F416, TVIPS
TemCam-XF416, Thermo Fisher Ceta (16M, 2, and D models),
Thermo Fisher Falcon III, and the Direct Electron DE-20. The
Falcon III and DE-20 are direct electron detectors; for these cases,
a beamstop was used to mask the incident beam at all times while in
diffraction mode to protect the sensor.
2.3 Image
Acquisition Software
In general, image acquisition software for the collection of
continuous-rotation MicroED data should be compatible with
both TEM and camera in order to script concerted actions for
automation. SerialEM natively supports diffraction mode and is
compatible with a wide range of microscopes and cameras. It also
includes a Low Dose Mode, which enables the user to customize
the camera settings of a set microscope lens configuration for a
given task. In this case, we will use different modes to set up (a) a
low-magnification crystal search by whole-grid atlas, (b) a Record
beam for data collection, and (c) a low-dose diffraction-defocus
beam (offset from the Record beam) [6, 7] for manually centering
the crystal as necessary.
3 Methods
3.1 Microscope
and Software Setup
A well-aligned TEM electron-optical system for imaging in the SA
magnification range is the ideal starting point for microscope alignment for the diffraction experiment [5, 8, 9]. Once switched to the
diffraction mode, the user selects the appropriate camera length,
adjusts dose (C2 intensity), and focuses the direct beam to a sharp,
condensed point via diffraction focus. Astigmatism of the diffraction beam should be checked at this point. The result is the diffraction beam used for data collection and is ultimately saved in the
SerialEM’s Low Dose Mode as the Record beam.
1. Start SerialEM. Make sure the phosphor screen is down and the
beam is visible.
2. Enable the Low Dose Mode by clicking its box under “Low
Dose Control”.
Automation of Continuous-Rotation Data Collection for MicroED
323