STANDARD OPERATING PROCEDURE JEOL JEM

STANDARD OPERATING PROCEDURE
JEOL JEM-2100 HR
TRANSMISSION ELECTRON MICROSCOPE
(TEM)
EDS Chemical analysis
Post column annular dark field detector
Tomography unit for 3D images
Cryogenic temperature control down to 77 K
Point Resolution:
Line Resolution:
STEM resolution:
Magnetic imaging resolution:
20 November 2010, R. Ubic
1
2.3 Å
1.4 Å
10 Å
22 Å
Emergencies
Power failure
The microscope will shut down safely. When power is restored, it is necessary to
restart the microscope manually (see below).
Cooling Water Suspension
The high voltage power and lens power will shut down, and the vacuum system will go
into the protected state. Upon resumption of cooling water, the microscope must be
restarted manually (see below).
Emergency Stop
Use the EM stop button (in left-hand cabinet) in case of fire or severe flood. Avoid this
method of shutdown unless absolutely necessary.
20 November 2010, R. Ubic
2
Start-Up Procedure
Preliminaries
Under normal circumstances, the microscope should be switched on and ready for use.
Check to make sure that SF6 gas pressure > 0.01 MPa (round dial gauge on left-hand
unit behind microscope).
Check to make sure that the air pressure is > 0.4 MPa (top round dial gauge behind grill
at base of column on left-hand side).
Check to make sure that the gun gas pressure is > 0.3 MPa (bottom round dial gauge
behind grill at base of column on left-hand side).
Check to make sure that the column pressure gauge on the sputter ion pump (SIP)
power supply is < 2.5 x 10-5 Pa (the rectangular dial gauge on the tall unit behind the
microscope).
Make sure that the tiny TEM icon on the taskbar of the left-hand monitor has a rotating
blue circle around it.
If any of these conditions are not met, contact Rick Ubic (ET 207, x62309) and do not
attempt to use the microscope.
20 November 2010, R. Ubic
3
Raising the Voltage
All references to the screen refer to the left-hand monitor unless otherwise stated.
If the “Controller for JEM-2100/HR” software is not currently running, double click on the
TEMCON icon on the desktop of the left-hand monitor.
The beam current should be zero and the high voltage (HT) switched off unless the
microscope has recently been used, in which case the HT will be left on and beam
current non-zero.
If the anti-contamination device heater is in place, carefully unplug it and remove it.
Click on the HT button at the top of the screen.
While you are waiting for the high-voltage to stabilize, fill the anticontamination device
(ACD) dewar with liquid nitrogen.
For operation at 100 - 200 kV:
Use the ▲ and ▼ buttons to increase the kV setting to 100 kV. The Step option allows
you to control the step size of the changes.
Press the HT ON button and wait for the voltage to stabilize at 100 kV.
If the desired voltage is greater than 100 kV:
Click on the More box to obtain more options.
In the Auto HT section, set the Target voltage to the desired value. Again, the
step controller will allow you to change the size of voltage steps.
Set the Step size to 0.1 and the Time/Step to 3.
Press the Start button in the Auto HT section. If the target voltage is 200 kV,
then this will take about 50 minutes.
Once the HT stabilizes, the beam current (top left of screen) should be about
half the voltage (in kV) µA (or about 101 µA at 200 kV). If it is > 105 µA at
200 kV, contact Rick Ubic or Steve Letourneau and do not attempt to use the
microscope.
20 November 2010, R. Ubic
4
Sample Insertion
Always wear gloves when handling specimen holders.
Single-Tilt Holder
Insert cartridge handling tool into hole at end of holder and open clamp by tilting the
tool. Carefully remove the cartridge from the holder with tweezers, lifting it over the
retaining pin.
Place the cartridge on the pale plastic holder, locating it on the pin.
Use the screw driver supplied to loosen the copper specimen retainer and rotate it clear.
If a specimen is already loaded (it should not be), remove it.
Place new specimen (facing upward) in the cartridge.
Return specimen retainer to its original position and secure it with the retainer screws.
Open the clamp on the holder again with the cartridge handling tool and re-insert the
cartridge, carefully locating it on the pin.
Double Tilt Beryllium Holder
Use the screwdriver supplied to loosen the screws on the copper retainers.
Very carefully lift them from the pins and rotate them clear of the beryllium wing-shaped
sample retainer.
Gently lift the sample retainer out and place it down on a clean part of the holder.
If a specimen is already loaded, remove it.
Insert new specimen (facing upward) in the holder.
Replace the beryllium sample retainer over the specimen.
Carefully maneuver the copper retainers back over the beryllium sample retainer and
gently screw them down.
Ensure the sample is being held securely in the holder by gently tapping – DO NOT HIT
THE HOLDER.
20 November 2010, R. Ubic
5
Inserting Specimen Holder into Microscope
See diagram.
Ensure that there is no dust or lint on the holder O-rings. If there is, wipe gently with
lint-free tissue.
Ensure that there is sufficient vacuum grease on the O-rings (they should be shiny but
have no noticeable white traces of grease). If excess grease is present, wipe it away
with lint-free tissue.
Align the specimen holder guide pin with the guide groove in the goniometer. Push the
holder into the goniometer until it stops.
Holding the specimen holder firmly in place, flip the goniometer switch to PUMP. The
yellow light should appear as evacuation commences.
Wait a few minutes for the green light to appear. DO NOT ROTATE THE HOLDER
YET. If using the double tilt holder, place the y-tilt control cable on top of the
goniometer for support. Do not let the cable dangle.
Turn the holder clockwise until it stops and insert the holder as far as it will go (not far).
Turn the holder again clockwise until it stops and insert the holder fully into the column
until it stops. The yellow light on the goniometer will go out at this point.
If using the double tilt holder, plug the y-tilt control cable into the TL-Y socket on the
goniometer.
Select the appropriate specimen holder on the top right of the Controller for JEM2100/HR main window:
Single-tilt holder, select EM-21010//21020: Single Tilt Holder.
Double-tilt holder, select EM-31640: Specimen Tilting Beryllium Holder.
Ensure that the SIP vacuum gauge recovers to < 2.5 x 10-5 Pa before continuing.
20 November 2010, R. Ubic
6
Filament Ramping
Make sure the Normal Setup button is selected in the Filament Control Speed.
The filament should be set to ramp to some percentage (typically 60% - 70%) less than
the maximum limit. Do not alter this number or the filament maximum limit.
Press the filament ON button in the High Voltage Control window (or the Beam button
on the top left of the left-hand control panel).
The Filament window appears. While it is open you cannot access any other windows.
Alignment Procedure
Finding the beam
The last user should have left the microscope at a magnification of about 50k and with
all the apertures out. If you can't find the beam, the most likely reason is that your
sample is in the way. Try moving it around. DO NOT move apertures or use beam shift
knobs to find the beam!
If you can't find the beam, try:
1. Check that the beam current has reached its normal value (½ kV ± 10%).
2. Check that all the apertures are out.
3. Check that the spot size is set to 1 (use SPOT SIZE knob on left-hand panel).
4. Decrease the magnification and spread the beam.
5. Go to LOW MAG mode (button on right-hand panel) and spread the beam with
the BRIGHTNESS knob (on left-hand panel).
6. Move your sample around with the trackball.
7. If you still can't find it, turn off the filament and remove the sample holder to the
park position. Turn on the filament again and repeat the above steps to find the
beam. If you find it, center it, then shut off the filament, re-insert your sample,
and begin again.
8. Try loading a previous alignment (Maintenance - Alignment - Load Alignment
File)
9. If you still can't find the beam, get assistance from Rick Ubic.
Gun Alignment
Find a hole in the sample.
For initial alignment, set the α-selector to 3. In general, for magnifications less than
≈100k, α = 3 is appropriate. Setting 2 is recommended for magnifications above 100k,
and setting 1 is recommended for magnifications of 400k and above.
20 November 2010, R. Ubic
7
Condenser Aperture Centering
Insert a condenser aperture.
Set the magnification to 100k.
Focus the beam with the BRIGHTNESS knob and center it with the SHIFT knobs.
Turn the BRIGHTNESS knob clockwise. If the illumination shifts, re-center it with the
condenser aperture X and Y knobs.
Repeat until the illumination no longer moves off center.
Gun Tilt Correction
Focus the beam.
Press F4 on the right-hand panel and use the DEF/STIG knobs until the beam is circular
and as small as possible. It may help to watch the current density value - the point is to
maximize the current density (brightness) on the screen.
Press F4 again to de-select the gun assignments.
Condenser Astigmatism Correction
Press the COND/STIG button (left-hand panel) and use the DEF/STIG knobs to
sharpen the emitter image. It may help to slightly desaturate the filament or increase
the spot size, keeping the image as small and circular as possible.
Press the COND/STIG button again to shut it off.
Condenser Lens System Alignment
Set SPOT SIZE (knob on left-hand panel) to 5
Focus the beam and center it using the beam SHIFT knobs.
Change SPOT SIZE to 1
Focus the beam and center it using GUN SHIFT (press F4 on right-hand panel then use
SHIFT knobs).
Repeat these steps until the beam remains centered. Finish with SPOT SIZE set to 1
and the beam spread on the sample.
20 November 2010, R. Ubic
8
Voltage Axis Centering
Try to find a pointed spot on the sample and center it
Press STD FOCUS button (right-hand panel) and focus on the sample using the Z
controls.
Adjust the magnification to about 100k
Finely focus the image using OBJ FOCUS.
Select Maintenance from the Controller for JEM-2100/HR software menu bar and click
on Alignment.
Click on the HT and CLA buttons in the Alignment Panel for Maintenance window.
Adjust the DEF/STIG knobs so that the image expands and contracts around the center
of the screen.
Click again on HT to shut this wobbler off.
Condenser Lens Deflection Coil Adjustment (Tilt Purity)
Focus the beam..
Click on Tilt in the Compensator portion of the Alignment Panel for Maintenance
window.
Click on TiltX in the Wobbler portion of the Alignment Panel for Maintenance window.
Unify the resulting two spots with the DEF/STIG X knob (left-hand panel). This is
sometimes easier with a slightly desaturated filament.
Click on TiltX again to turn it off.
Click on TiltY in the Wobbler portion of the Alignment Panel for Maintenance window.
Unify the resulting two spots with the DEF/STIG Y knob (right-hand panel). This is
sometimes easier with a slightly desaturated filament.
Click on Tilt in the Compensator portion of the Alignment Panel for Maintenance
window to turn it off.
20 November 2010, R. Ubic
9
Shift Purity Adjustment
Press SA DIFF (right-hand panel) and set the camera length to 200cm with the
MAG/CAM L knob on right-hand panel.
Turn the BRIGHTNESS knob fully clockwise.
Use DIFF FOCUS knob (right-hand panel) if you cannot see the edge of the beam.
Use the DIFF FOCUS knob (right-hand panel) to reduce the caustic spot. You may find
it useful to then underfocus slightly.
If the caustic spot moves off the screen center as the BRIGHTNESS knob is turned,
center it using the DEF/STIG knobs (but make sure the BRIGHT TILT button is
illuminated).
Click on Shift in the Compensator portion of the Alignment Panel for Maintenance
window.
Click on ShiftX in the Wobbler portion of the Alignment Panel for Maintenance window.
Unify the resulting two spots using the DEF/STIG X knob.
If the spot moves off-screen, reduce the camera length and continue adjusting
If the spot splits into two in the Y direction, click on Compensator Angle then
unify the spot with the DEF/STIG X knob. Click on Angle again to turn it off.
Click on ShiftX again to turn it off.
Click on ShiftY in the Wobbler portion of the Alignment Panel for Maintenance window.
Unify the resulting two spots using the DEF/STIG Y knob.
If the spot moves off-screen, reduce the camera length and continue adjusting
If the spot splits into two in the Y direction, click on Compensator Angle then
unify the spot with the DEF/STIG X knob. Click on Angle again to turn it off.
Click on ShiftY again to turn it off.
Iterate the Tilt Purity and Shift Purity alignments until both are simultaneously aligned.
20 November 2010, R. Ubic
10
Objective Lens Astigmatism Correction
Find the amorphous edge of the sample and increase magnification to ≈300k - 500kX,
adjusting the BRIGHTNESS knob for optimum viewing conditions and using the ShiftX
and ShiftY knobs to center the beam (ensure that the BRIGHT TILT button is
illuminated).
Finely focus with the OBJ FOCUS knobs.
Press the OBJ STIG button (left-hand panel)
Adjust the DEF/STIG knobs until minimum contrast is observed in the amorphous
region. It may help to slowly turn OBJ FOCUS back and forth through focus to observe
the variation of phase contrast.
Press the OBJ STIG button again to shut this adjustment off.
Focusing
If an objective aperture is in place, remove it.
For low magnifications (< ≈100kX), focusing can be done easily with the image wobbler.
Press the IMAGE WOBB X and/or IMAGE WOBB Y button (right-hand panel).
Unify the oscillating image with the OBJ FOCUS knobs (right-hand panel). If the
image cannot be unified, try correcting for objective astigmatism (above).
Press the IMAGE WOBB X and/or IMAGE WOBB Y button again to shut it/them
off.
For high magnifications (> ≈100k X), focusing is best done with either Fresnel fringes or
point images.
Look for the appearance of bright and/or dark Fresnel fringes. If they are very
close together, their appearance results in an increase in contrast.
For widely-spaced fringes, use the Z control to bring them together. Only use
OBJ FOCUS knobs to correct for very small fringe spacings.
For very high magnification (> 300k X), find an amorphous section of the sample
(typically near the edge) and focus the image using the OBJ FOCUS knobs. The
point image will have minimum contrast at the point of focus.
Note: The optimum focal condition is not always at exact focus. It is often useful to
slightly underfocus samples, especially biological ones, in order to increase contrast.
20 November 2010, R. Ubic
11
Obtaining a Selected Area Diffraction Pattern (SADP)
Insert a Selected Area Aperture (aka Field Limiting Aperture). It is recommended that
the largest one possible which still excludes extraneous parts of the sample be used.
Use the X and Y positioning knobs on the aperture to center it on the region of interest.
Press the SA DIFF button (right-hand panel). An image of the back focal plane should
appear.
Adjust the MAG/CAM L knob (right-hand panel) to the desired camera length (120cm is
recommended for viewing but 25cm for recording an image).
The intensity of the pattern can be adjusted with the BRIGHTNESS knob.
Adjust the DIFF FOCUS knob (right-hand panel) to obtain the sharpest pattern.
Center the pattern by pressing PLA on the left-hand console and adjusting position with
ShiftX and ShiftY knobs.
To record the pattern, use a small camera length (≈40 cm) and ALWAYS USE THE
BEAM STOP to block the central transmitted beam (failure to do this can cause
irreparable damage to the camera). See “Recording Images on the Orius SC1000
Digital Camera” below for more information.
Kinematical Bright-Field (BF) Imaging
Obtain an SADP as described above.
Insert an objective aperture and use the X and Y adjustment knobs to center it over the
transmitted beam. The smaller the aperture, the more contrast will be in the image, but
also the less intensity and the more potential for aberrations.
Press MAG1 (right-hand panel) to return to image mode.
20 November 2010, R. Ubic
12
Scanning Transmission Electron Microscopy (STEM)
Focus the beam with the Brightness knob.
Make sure the BRIGHT TILT button is illuminated and move the beam to the center of
the screen using the ShiftX and ShiftY knobs.
Click on the Dialogue option in the TEMCON menu bar, then select ASID Control.
The axis alignment procedure is not normally necessary. If desired resolution cannot be
attained, contact Rick Ubic or Steve Letourneau.
1. Click on the Alignment radio button in the Function section of the ASID
Control Panel window.
2. Click on the Spot Size arrow buttons so that an appropriate spot can be
obtained.
3. Click on the Spot Mode icon near the top of the image Control window.
4. Move the cross cursor to the center of the screen by clicking on the Center
button in the Image Control window.
5. Turn the BRIGHTNESS knob fully clockwise.
6. Obtain a caustic spot using the OBJ FOCUS knobs.
7. Move caustic spot to the center of the screen using DEF/STIG knobs.
8. Adjust the BRIGHTNESS knob to obtain an image, press the STD FOCUS
button (right-hand panel), then focus the image with OBJ FOCUS.
9. Focus the beam with the BRIGHTNESS knob.
10. Center this crossover spot using the ShiftX and ShiftY knobs.
11. Repeat steps 5 - 10 until the caustic spot and crossover image coincide with
the screen center.
Open Simple Image Viewer (icon on desktop).
Click on the MAG radio button in the Function section of the ASID Control Panel
window. If the beam is not in the center of the screen, carry out the axis alignment
procedure described above.
Click on the Scan Mode icon in the Image Control window.
20 November 2010, R. Ubic
13
Click on the STEI-BF radio button in the ASID Control Panel window.
Click on the Spot Mode icon in the Image Control window.
Remove the condenser aperture to observe the Ronchigram pattern (this is more visible
in an amorphous sample).
If the Ronchigram is small and difficult to see, increase the camera length by
using the Camera Length arrow buttons in the Mag/Camera length section of the
ASID Control Panel window.
If you cannot obtain a Ronchigram, adjust the OBJ FOCUS knobs accordingly.
If the Ronchigram is off-center, move it to the center by pressing the PLA button
(left-hand panel) and adjusting the DEF/STIG knobs.
If the Ronchigram is not circular, or if adjusting the OBJ FOCUS knobs make it elliptical,
press the COND STIG button (left-hand panel) and adjust the DEF/STIG knobs
accordingly. Press the COND STIG button again to switch it off.
Insert condenser aperture 3 and adjust it so that its center coincides with the center of
the Ronchigram pattern.
Insert an objective aperture and adjust it so that its center coincides with the center of
the Ronchigram pattern.
Insert the JEOL camera (left of column above viewing chamber) and cover the viewing
window.
To correct for astigmatism:
Slightly defocus a STEM image.
If the image appears unidirectionally blurred, press the COND STIG button (lefthand panel) and adjust the image with the DEF/STIG knobs.
Refocus the image.
Repeat until no astigmatism is observed.
20 November 2010, R. Ubic
14
Tomography
Insert the holder and bring up the beam at 3000x. Make sure the beam is spread out but
don't go all the way to fully overfocus with CL3.
Acquire a trial image with SerialEM. If what you see looks contrast-rich and has many
features, start 'eucentric - rough' from the task menu.
Once this completes, locate your ROI and set the magnification and brightness (you can
move the stage and set the brightness all through SerialEM).
Once you have an ROI for tomography run 'eucentric - fine' from the task menu.
Walk up the stage to 53 degrees using 'walk up' from the task menu. This starting angle
may be higher if you use a High Tilt Retainer and no in-gap OL aperture in the beam
path.
From the 'tilt series' menu choose 'Setup/start' to evaluate your tomography parameters.
Make sure that the angles are properly specified and pick your basic increment (1.5 or 2
is perfectly fine, or if you want to get more detail in the tomogram you can pick 1
degrees). Pay attention to the mag and the 'at start and when needed track at
magnification'. Rule of thumb here is the latter should probably be ~1/3rd of the imaging
magnification.
Keep the beam intensity for the moment constant.
Most biological samples do fine with a focus of -0.35 micron, so do most materials
samples. For cryo you need more, say 3-6 microns. If the ROI has features that only
show with a lot of defocus, you can add this as an offset to the autofocus (remember
that underfocus is negative!).
For initial action you probably should have a trial image in focus before you bring up this
window, so 'align to image now in Buffer A' should be on.
Leave tracking unchanged for the moment.
Hit 'GO' and sit back. Remember there are 'End' and 'STOP' on the camera and macro
controls panel that will halt the tilt series acquisition.
Once the series is done, terminate it from the 'Tilt Series' menu and close the file and do
not leave 90° rotated copies in various buffers.
You can run up the tomogram in etomo.
20 November 2010, R. Ubic
15
End of Session
Set magnification to ≈50,000X.
Spread illumination to fill the screen.
Double click on the Stage Neutral button
Remove all apertures.
Turn off the filament by pressing OFF in the Filament section of the High Voltage
Control window (press the HT button at the top of the screen if this window is not still
open).
Wait until filament is completely off before extracting the holder. Do NOT remove the
holder while the filament is turning off.
Remove sample from holder. DO NOT STORE SAMPLES IN MEC113/113a.
Record hours in the log book.
Cover the viewing chamber.
If you are the last user of the day, insert the ACD heater (watch out for excess nitrogen
that may pour out of the tube) and plug it in, then turn on the ACD heat by clicking on
Maintenance, ACD & Bake, and pressing ON in the ACD Heat tab.
20 November 2010, R. Ubic
16