3.2.1 Electron
Microscope Checkup
1. Several TEM routines are needed to be carefully checked and
tuned before the experiments.
(a) Check the liquid nitrogen levels in the cold-trap. If the
liquid nitrogen levels are low, fill up the liquid nitrogen
and wait until the stage is stable.
(b) Check the TEM vacuum status if the pressures are in a
normal range.
(c) Make sure that the stage is pre-cooled to liquid nitrogen
temperatures.
(d) Open the column valve and see if a stable beam can be
found in the low magnification.
(e) Check if the digital camera is cooled down.
(f) Prepare gain and dark references for the digital recording
camera.
2. If cryo-specimen transfer holder will be used for grid transfer,
several routines are needed to be prepared:
(a) Before using the cryo-specimen transfer holder, check if
there is any residual grid or contamination on the tip.
Remove them before cooling the holder.
(b) Clean up and check if there is any residual grid or contamination in the transfer station before cooling down.
(c) Cool down the holder and the transfer station with liquid
nitrogen to À180
C and wait until it is stable.
(d) Use fresh liquid nitrogen to cool down the holder and
transfer the grid specimen.
3.2.2 Beam Alignment
1. Open the column valve. Find the beam on the screen at low
magnification. Adjust the beam intensity and centered the
beam by varying beam intensity/brightness (C2 lens current)
and beam shift. Keep the objective aperture and selected area
diffraction (SAD) aperture out of the beam path at this stage.
2. If the specimen grid is inserted, find an area without any carbon
film or specimen.
3. Align the beam conditions for electron diffraction or imaging.
Turn on the Low Dose system and switch to the Exposure
mode. Keep the mode changing in a sequence from the Search,
Focus, and Exposure. First set the magnification to about
5000Â.
4. Align the gun tilt to maximize the brightness and align the gun
shift according to different spot sizes (C1 lens).
5. Choose the target spot size for electron imaging or diffraction
(see Note 11).
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Ka-Yi Chan et al.
Microscope Checkup
1. Several TEM routines are needed to be carefully checked and
tuned before the experiments.
(a) Check the liquid nitrogen levels in the cold-trap. If the
liquid nitrogen levels are low, fill up the liquid nitrogen
and wait until the stage is stable.
(b) Check the TEM vacuum status if the pressures are in a
normal range.
(c) Make sure that the stage is pre-cooled to liquid nitrogen
temperatures.
(d) Open the column valve and see if a stable beam can be
found in the low magnification.
(e) Check if the digital camera is cooled down.
(f) Prepare gain and dark references for the digital recording
camera.
2. If cryo-specimen transfer holder will be used for grid transfer,
several routines are needed to be prepared:
(a) Before using the cryo-specimen transfer holder, check if
there is any residual grid or contamination on the tip.
Remove them before cooling the holder.
(b) Clean up and check if there is any residual grid or contamination in the transfer station before cooling down.
(c) Cool down the holder and the transfer station with liquid
nitrogen to À180
C and wait until it is stable.
(d) Use fresh liquid nitrogen to cool down the holder and
transfer the grid specimen.
3.2.2 Beam Alignment
1. Open the column valve. Find the beam on the screen at low
magnification. Adjust the beam intensity and centered the
beam by varying beam intensity/brightness (C2 lens current)
and beam shift. Keep the objective aperture and selected area
diffraction (SAD) aperture out of the beam path at this stage.
2. If the specimen grid is inserted, find an area without any carbon
film or specimen.
3. Align the beam conditions for electron diffraction or imaging.
Turn on the Low Dose system and switch to the Exposure
mode. Keep the mode changing in a sequence from the Search,
Focus, and Exposure. First set the magnification to about
5000Â.
4. Align the gun tilt to maximize the brightness and align the gun
shift according to different spot sizes (C1 lens).
5. Choose the target spot size for electron imaging or diffraction
(see Note 11).
258
Ka-Yi Chan et al.