3.2 Cryo-Transfer
(See Note 4)
1. Cool cryo-transfer station with liquid nitrogen and transfer
sample grid box containing sample grids into the transfer station sample platform.
2. Having followed procedures for preparation of the 626 cryotransfer holder—in particular regeneration of the zeolite in the
holder dewar—remove the holder from the 655 pumping station following manufacturer’s instructions and place into the
cryo-transfer station.
3. Fill sample holder dewar with liquid nitrogen and refill transfer
holder station with liquid nitrogen to a level above the holder
rod. Pull back protective copper blades to allow sample placement onto holder.
4. Remove grid from sample box and place onto sample holder
with pre-cooled fine tip tweezers.
5. Place copper clip ring on top of grid and secure onto sample
holder. Cover sample with protective blades.
6. Remove transfer holder from station and place into microscope
CompuStage at the correct orientation, making sure to minimize the amount of time the holder spends in air.
3.3 Grid Screening
and Preliminary
Diffraction
1. Assuming operation of a fully calibrated instrument in microprobe mode, perform eucentric height alignment by wobbling
the sample and adjusting the z-height.
2. Focus the system on the back focal plane of the objective lens
by adjusting the focus knob at a magnification greater than
25,000 Â while viewing the edge of a hole or a distinctive
feature on the carbon support.
3. Expand the beam using the intensity knob to a near parallel
state, and set spot size to 11 (see Note 8).
4. Press diffraction button on console to operate the microscope
in diffraction mode. Ensure the beam is free of astigmatism (see
Note 9).
5. Position the beam at the center of the detector and calibrate the
location of the selected area aperture (SAA) as needed.
6. For sample detection, expand the beam by adjusting the power
to the condenser lens used to focus the beam. This can be
achieved, for example, by turning the intensity knob to enter
overfocused diffraction mode. One could pre-set this configuration using low-dose settings. Scan the grid for crystals by
moving the stage x, y location using the trackball or joystick.
If available, low-dose mode can automate the focus adjustments to minimize electron exposure on samples while the
grid is examined. Where low dose is available, visual inspection
of the grid can be performed in overfocused diffraction under
MicroED of Small Macromolecules
335
(See Note 4)
1. Cool cryo-transfer station with liquid nitrogen and transfer
sample grid box containing sample grids into the transfer station sample platform.
2. Having followed procedures for preparation of the 626 cryotransfer holder—in particular regeneration of the zeolite in the
holder dewar—remove the holder from the 655 pumping station following manufacturer’s instructions and place into the
cryo-transfer station.
3. Fill sample holder dewar with liquid nitrogen and refill transfer
holder station with liquid nitrogen to a level above the holder
rod. Pull back protective copper blades to allow sample placement onto holder.
4. Remove grid from sample box and place onto sample holder
with pre-cooled fine tip tweezers.
5. Place copper clip ring on top of grid and secure onto sample
holder. Cover sample with protective blades.
6. Remove transfer holder from station and place into microscope
CompuStage at the correct orientation, making sure to minimize the amount of time the holder spends in air.
3.3 Grid Screening
and Preliminary
Diffraction
1. Assuming operation of a fully calibrated instrument in microprobe mode, perform eucentric height alignment by wobbling
the sample and adjusting the z-height.
2. Focus the system on the back focal plane of the objective lens
by adjusting the focus knob at a magnification greater than
25,000 Â while viewing the edge of a hole or a distinctive
feature on the carbon support.
3. Expand the beam using the intensity knob to a near parallel
state, and set spot size to 11 (see Note 8).
4. Press diffraction button on console to operate the microscope
in diffraction mode. Ensure the beam is free of astigmatism (see
Note 9).
5. Position the beam at the center of the detector and calibrate the
location of the selected area aperture (SAA) as needed.
6. For sample detection, expand the beam by adjusting the power
to the condenser lens used to focus the beam. This can be
achieved, for example, by turning the intensity knob to enter
overfocused diffraction mode. One could pre-set this configuration using low-dose settings. Scan the grid for crystals by
moving the stage x, y location using the trackball or joystick.
If available, low-dose mode can automate the focus adjustments to minimize electron exposure on samples while the
grid is examined. Where low dose is available, visual inspection
of the grid can be performed in overfocused diffraction under
MicroED of Small Macromolecules
335