CM 200 Instructions 1
Asturias Lab Instructions for CM200
Last updated October 2004
SOME GENERAL THINGS TO REMEMBER:
The day before imaging
1. Fill up two liquid nitrogen dewars (ice crystals will settle to bottom of the dewar if the
nitrogen sits overnight, thereby reducing the chances of sample contamination)
2. Place cryostage into the pumping station. Consecutively open all the valves (4 total, start
with the one closest to the turbo pump, then open the two butterfly valves, and last open
the valve on the cryostage).
While working on the microscope
1. Be VERY careful with the cryostage; never bounce into it. Be especially careful
when adding liquid nitrogen.
2. The objective aperture needs to be taken out temporarily during alignment. Make
sure it is back in before taking pictures.
3. At any time when the beam is not required (filling up the dewars, taking a break,
developing film, etc) close the gun valve and sample shield (small knob on the
cryostage handle should be pushed towards the column). Open and close gun
valve VERY slowly, or the vacuum system will sense a disturbance and shut
down.
Before taking pictures, check
1. Objective aperture (it should be in, i.e., turned to the left, pointing away from the
door)
2. CM/CCD mode switch should be in CM if you intent to collect imag es on film)
3. Nitrogen level in cryostage and cold trap dewar.
4. Eucentric height of the specimen (Z-height, use A-axis wobbler and minimize
sweeping movement of specimen)
5. Check exposure time and the size and position of the beam in exposure mode
While taking pictures check:
1. Eucentric height of the specimen (every time you change squares on the grid)
2. Exposure time and exposure area (often during first 15 minutes, then
approximately every 10-15 pictures)
3. Nitrogen level in cryostage and dewar (approximately every 30 minutes)
CM 200 Instructions 2
GENERAL INFORMATION
We operate the microscope in TEM
BRIGHT-FIELD -LOW DOSE, which
facilitates imaging previously
unexposed areas of a beam-sensitive
specimen. Within the Low-dose there
are three individual modes that we
use (settings are independent for
different modes):
SEARCH MODE
Function: checking a sample under
low magnification and low electron
dose conditions to identify areas
suitable for imaging.
Magnification: 2450x
Spotsize: usually 3-4; the largest
value (to lower beam intensity and
thus minimize damage) that allows
good overview of specimen areas of
interest.
EXPOSURE MODE
Function: data collection.
Magnification: 58kx, which equals
66kx when the screen is lifted.
Spotsize: usually 5 (in general
between 4-7), choose it so that the
required exposure time is between
0.5 – 1.0 s. Then set the manual
exposure time within 20% of the
required exposure time (manual exposure time can only be set to certain predetermined values.
Remember that it is always better to have slightly underexposed pictures, as they are easier to
scan).
FOCUS MODE
Function: focusing on areas of the specimen adjacent to the area selected for data collection.
Magnification: 135kx
Spotsize: 4-6
Positions for S1 and S2: 180 degrees apart, and 2.5-3.0 μm apart from image center.
Focus step size used for focusing: 4, 3 (rarely 5-7 when very far from focus).
Focusing:
Focus first at one (S1 or S2) position. Remember that when overfocused the carbon film
support grains appear dark. Underfocused grains appear light. To focus find value of
minimum contrast which is in between overfocused and underfocused values.
Press RESET DEFOCUS button, then focus again using S2.
The mean value between SI and S2 is used as the optimal focus value from which you defocus
(usually we underfocus -0.386 um up to -1.96 um).
See Troubleshooting if you have problems focusing.
CM 200 Instructions 3
TRANSMISSION ELECTRON MICROSCOPE (TEM)
Ray diagram for a complete electron microscope. Filament F, condenser 1 lens C1, condenser
2 lens C2, condenser aperture CA, specimen S, objective lens O, objective aperture OA (in the
back focal plane). 1st intermediate image and selector aperture SA. Intermediate lens PI,
second intermediate image h, projector lens P2 and final image on the fluorescent screen F.
CM 200 Instructions 4
OPERATION PROCEDURES
PREPARATION
1) PRESTART
Fill thermo-cold dewar for cold trap with liquid nitrogen up to the top, and cover the dewar
with the Styrofoam cover to avoid nitrogen evaporation and formation of ice. Record the
values of P3 (Vacuum page on scope, normal value is between 30 and 80), room
temperature (usually ~ 68F), and relative humidity (usually ~ 68%) into the EM logbook.
2) CHECK VACUUM STATUS
Check that UV and HVAC lights (located on right side of control panel), if HVAC light is not
on check that the camera chamber is closed or see if film has been changed recently (TEM LOW DOSE->PARAMETERS
Set "GUN LENS" to 3
Set "VOLTAGE" to 120 kV
Turn High Tension ON (hold down "HIGH TENSION" button on console for ~1 second)
Go to Configuration Page MODES->CONFIGURATION->DISPLAY check extraction
voltage limit, it should be 3.81. DO NOT SET HIGHER THAN THIS VALUE!
Turn FILAMENT KNOB slowly clockwise until it reaches the extraction limit of 3.8kV
(actually 3.81). "STANDBY" will change to "OPERATION" as soon as the filament knob is
turned.
Slowly open GUN VALVE. Check to see if you can find the beam at low mag (~2500X). If
no beam is present go to lower magnification and adjust the beam shift. After finding the
beam close GUN VALVE and proceed.
4) LOADING THE SPECIMEN
4.1) LOADING the specimen into the cryostage
Grease holder’s O-ring (ONLY USE WHITE FOMBLIN grease, that can be found in a
syringe on the desktop, or the drawer in the F20 room). Put a very small amount of
grease on your finger, and rub it on the o-ring on the cryostage rod. Use only enough
grease to leave make the o-ring look shiny, NOT MORE.
Remove clip ring and old sample from rod tip PRIOR TO COOLING!
Connect cryo-holder to the temperature meter, and make sure that heater is off (panel
with the current reading is not lit). Add liquid nitrogen to cryo-holder’s dewar and make
sure temperature of tip starts going down (may take a minute). As soon as temperature
starts to go down, add LN2 to sample tip (BEFORE it is cold enough to allow moisture to
condense and contaminate the tip). Wait until the tip is at -160 C. Load the specimen.
4.2) LOADING the specimen into the MICROSCOPE
CM 200 Instructions 5
Make sure that the gun valve is CLOSED.
For cryo samples set the goniometer tilt to -55. This is accomplished using the Tilt-
knob on the left panel (clockwise is ‘+’, counterclockwise is ‘-‘, it works as ‘gas pedal’, the
more you turn knob, the faster the value changes, turn the knob to original position when
the correct value is reached) or by loading one of the saved positions from the
compustage page.
Press VACUUM ON (right panel) in order to pre-pump airlock before inserting the
specimen.
Move the mounting station with the cryostage to the microscope. Pull the cryostage out
of the mounting station, and turn it 90 º CW, so that the small pin on the top of the tip is
at the 3 o’clock position. Insert the specimen holder into the airlock entry and slide it in
until a stop is reached. A RED airlock indicator LIGHT will turn on. Wait for pumping.
When the red light goes off, press RESET AB on the Compustage page, wait while the
stage rotates, then rotate the holder 90º CCW and allow it to slide slowly into the
column. Maintain a firm grip on the holder (hold on to the back end of the rod sticking out
of the dewar) during insertion so that the holder does not enter the column too quickly
(due to the suction by the vacuum) and that no damage to goniometer and/or the holder
can occur.
Allow 10-15 minutes for vacuum to recover, and for cryostage to reach thermal
equilibrium. Attempting to examine the specimen too soon will result in specimen
contamination, and it will not be possible to collect any images, as the specimen will be
drifting considerably.
MICROSCOPE ALIGNMENT
1) CHECK COLUMN OF CM200
ALL APERTURES should be set on 4 (Except Diffraction Aperture, which is not used and
must be placed in OUT position (turned to the right, towards the door).
SLOWLY open the GUN VALVE, open the sample shield, go to SEARCH MODE, and find
an empty area.
2) SET MODES
Search mode: set magnification to 2450X
Exposure mode: set magnification TEMPORARILY to ~25KX
Focus mode: set magnification to 135X
If you cannot find the beam in focus mode, for each focus position, lower the magnification
until the beam appears on the screen, center with the SHIFT X/Y knobs (beam deflectors),
and go back to 135KX
Check FOCAL POINTS: go to focus SI or S2
SI: LDrad (3.00 um) LD rot (325º)
S2: LDrad (3.00 um) LD rot (145º)
The focal points should be about 3um away from center of exposure area, and
separated by 180º
LDrad is controlled by MULTIFUNCTION knob Y (linear image shift).
CM 200 Instructions 6
LDrot is controlled by MULTIFUNCTION knob X (rotational image shift).
3) CENTER THE CONDENSER APERTURE
Go to EXPOSURE mode (~25KX). Go to crossover (smallest bright spot) and center the
beam with the BEAM DEFLECTORS (SHIFT X/Y). Expand beam by turning INTENSITY knob
clockwise. Remember that you always want to operate in the region where the beam expands
when you turn the INTENSITY knob CLOCKWISE.
If condenser aperture is not centered, the circular beam will not be centered as you increase its
radius. Go to crossover and center the beam with SHIFT X/Y. Increase the size of the beam
until it is about the size of the big circle on the screen and center it by moving the condenser
aperture with the controls on the aperture mount. Go back to crossover and re-center the beam
using the SHIFT X/Y knobs. Repeat until the beam is centered at crossover and expands to
cover the screen uniformly.
NOTE: If the beam is not circular, press the STIGMATOR alignment knob, and adjust the
CONDENSER stigmators until the beam is circular.
4) ADJUST EUCENTRIC HEIGHT
To adjust the height of the sample go to SEARCH MODE, expand the beam, find a
recognizable feature, and center it on the screen. Press COMPUSTAGE to switch over to
the Compustage Register Control page, then press A-WOBBLER (Angle or A axis-wobbler)
- the specimen is now being tilted continuously from +15 degrees to -15 degrees, back and
forth.
Minimize image movement using the Z-joystick (if z-movement does not work, activate Z-
joystick on Compustage Control page). Pay attention to the movement of the compustage
and the image. The image will jump a little when the direction of motion changes but should
be steady in between, and the recognizable feature should stay in the same place. When
this is accomplished, key A-WOBBLER again to stop sweeping motion.
Go to EXPOSURE MODE and repeat the process for fine adjustments. At this point the
specimen should be set to the right height, and you may want to press the AUTO FOCUS
knob to get close to the correct FOCUS value.
3) ALIGNMENT OF THE GUN
Before starting check to make sure that you are adequately close to FOCUS!
Gun Shift Alignment
Go to EXPOSURE MODE, where the magnification should be set between 25K and
55K. Dim the lights in the room.
Press the ALIGNMENT button and key GUN SHIFT
Select spot size 9, and use the INTENSITY knob to bring the beam to crossover. If you
can’t see the beam, go down in spot size until you can see the beam, and then center it
using SHIFT X/Y. If necessary, go back up to spot size 9 and center the beam again
with SHIFT X/Y.
Select spot size 1, bring the beam to crossover, and center it with MULTIFUNCTION
X/Y knobs (beam shift should be highlighted in the alignment page).
Repeat the two steps above until the beam remains centered when you go back and
forth between spot sizes 1 and 9.
Gun Tilt Alignment (55k MAG / Spot-Size=1)
The correct gun tilt alignment will give a small, very intense spot in the center of the
crossover beam.
CM 200 Instructions 7
PRESS GUN TILT on the Alignment page.
Bring the beam to crossover; and you will see the bright spot. Ideally it would be in the
center of the crossover beam, but usually you have to adjust its position. Adjust gun tilt
slowly with the MULTIFUNCTION X/Y knobs until the bright spot is centered. When you
reach the edge of the screen, center spot using GUN SHIFT and MULTIFUNCTION X/Y
knobs. Repeat until the bright spot is perfectly centered. Exit Alignment page.
NOTE: Gun Alignment will change and need to be re-adjusted if the high tension setting
or the extraction voltage are changed.
6) ADJUST ROTATION CENTER
Function: Aligns the objective-lens rotation center to minimize image displacement as the
objective lens’ current is modulated.
Magnification ~50K (EXPOSURE MODE). Choose the same spot size as you plan to use
for taking pictures (usually 5-6).
NOTE: Intermediate magnifications in SA or LM range have different rotation centers. Also,
rotation center changes when you change stop size.
In SEARCH mode, find a recognizable feature on the sample and center it on the screen.
Bring the beam to crossover and check to make sure that you are close to focus.
Select ROT-CENTER on ALIGNMENT Selection page
Select FOCUS STEP 5 or 6 (this step size controls the amplitude of motion). Using the
MULTIFUNCTION X/Y knobs, minimize image movement so that it will move in and out (like
a cork in the sea), but not up/down or left/right.
7) ADJUST PIVOT POINTS
The alignment of the pivot points determines the relationship between two deflection coils
used. You want to make sure that the beam pivots around the correct point. Adjust in
EXPOSURE mode with the spot size the same as you plan to use for taking pictures
(usually 5-6).
Take the OBJECTIVE APERTURE OUT.
Press ALIGNMENT BUTTON (on the right of TEM control panel).
Press PIVOT POINT X.
The two circular beams need to completely overlap, to achieve this use the X/Y
MULTIFUNCTION knobs.
Repeat for PIVOT POINT Y. If significant adjustment was needed, re-check the rotation
center adjustment.
VERY IMPORTANT: put the OBJECTIVE APERTURE BACK IN (positioned to the left). This
is essential to provide contrast when imaging unstained specimens.
8) CENTER OBJECTIVE APERTURE
In SEARCH mode find an area with thick ice or some other diffracting material. Go to
EXPOSURE mode
CM 200 Instructions 8
Press D BUTTON (diffraction mode button) on right panel.
You should see a dim halo around the central bright spot. If necessary, adjust camera
length (MAGNIFICATION) or INTENSITY to make the halo more clear. Then, center the
halo around the small bright circle using the OBJECTIVE APERTURE centering knobs on
the aperture mount.
Press D BUTTON again to exit diffraction mode and spread the beam.
9) CORRECTING CONDENSER STIGMATOR (BEAM ASTIGMATISM)
Function: to make the beam circular.
NOTE: This procedure is usually done throughout the alignment when the beam becomes
non-circular, but it needs to be repeated with precision when the microscope has been
aligned. Also, note that stigmator settings vary from one mode to another and between
various spot sizes, so you need to correct it separately in each mode you use.
Press STIG BUTTON on the right-hand side of the control panel. This opens the
STIGMATOR control page.
Select COND (condenser lens). Make sure not to inadvertently change the settings for the
objective stigmators, which will be adjusted next.
Use MULTIFUNCTION X/Y knobs to correct for astigmatism until the beam is circular and
remains circular when it is being spread or condensed with the INTENSITY knob. After the
beam is circular exit the STIG page.
10) CORRECTING OBJECTIVE STIGMATOR (IMAGE ASTIGMATISM) WITH CCD CAMERA
Turn switch on the left of the microscope into the CCD camera position.
Turn computer monitor on and start the CCD camera program.
Before taking pictures with the camera, create a FLATFIELD to correct the CCD. Find an
empty square (no carbon), go to EXPOSURE mode, lift the screen, and unblank the beam.
On the camera dialog box menu click on Flatfield –> Create Flatfield –> Store Flatfield.
To take a picture: find an appropriate spot, focus, defocus (to desired underfocus value),
go to EXPOSURE MODE, lift screen, unblank beam, and press EXPOSURE BUTTON on
the camera dialog box menu. Exposure time is usually set to 500ms, but shorter times can
also be used (e.g., 300ms).
To check the power spectrum of an image click Power Spectrum on the camera dialog box
menu.
To correct objective stigmator: Find an area where the carbon film/ice look good. Focus,
and then underfocus about 1um. Go to EXPOSURE mode. Press the STIG button, and
select OBJ. On the camera dialog box menu select Continues Picture mode, displaying the
Power Spectrum. Adjust OBJ STIGMATOR with MULTIFUNCTIONAL X/Y knobs so that
Power Spectrum looks as circular as possible. Go to lower defocus (~200nm) to adjust
OBJECTIVE STIGMATOR with higher precision. In the end Power Spectrum should be
perfectly symmetric and circular.
CM 200 Instructions 9
11) FINAL CHECK OF THE LOW-DOSE MODE
Check the settings in LOW DOSE mode one last time to make sure that they are correct.
The main thing to check is the magnification in EXPOSURE more (60KX), the manual
exposure time (should match the meter reading for an area that has ice of the right
thickness), and the size and centering of the beam in exposure and focus modes.
TAKING PICTURES ON FILM
Areas where the ice thickness and quality are adequate should first be identified using the CCD
camera. It is a good idea to scan the entire grid in SEARCH mode, collect CCD images to
check for particles, and record in the compustage memory the positions (x,y, and eucentric
height) of squares where the particles look good. NOTE: To get adequate contrast in SEARCH
mode, the focus value in this mode should be set to about -60μm!!
IMPORTANT: Before switching to film, make sure the switch is in TM position (not CCD!!).
If you forget to set the switch properly, your pictures will be BLANK!
1) Find a good spot in SEARCH mode. Check eucentric height.
2) In EXPOSURE mode check that the beam is centered on the four corners of the film outline,
and that the exposure meter time is adequate (between 0.5 – 1 s) and within 20% of the
manual exposure time setting.
NOTE: Pay attention to whether you are overexposing or underexposing it will be important
when you develop film (see note on p.1)
3) FOCUS at focus step 4, 3 in S1 and S2 positions. Set focus to zero (press RESET
DEFOCUS)
4) UNDERFOCUS (‘—‘) turn counterclockwise to get to the needed value (usually between –
0.78 um – 1.92 um).
5) LIFT the fluorescent screen.
6) EXPOSURE BUTTON light comes on. Wait until everything has calmed down before
carefully pressing it. The panel light goes off, sit quietly, listen to film fall into the receiving box
and look for exposure light to come on. When panel lights come on put the SCREEN down.
7) Repeat.
REMEMBER TO CHECK THE FOLLOWING
Eucentric height of the specimen
At least every time when changing squares on the specimen grid
Exposure time and exposure beam position
Often during first 15 minutes, then approximately every 10-15 pictures
Nitrogen level
In cryostage and dewar (approximately every 30 minutes)
SHUTING DOWN MICROSCOPE TO CHANGE FILM
1) CLOSE gun valve and specimen shield, and set FEG to STANDBY mode
2) TURN OFF high tension
3) EXCHANGE film cassette
Go to adjacent room and open nitrogen gas valve.
Go to the Vacuum page. Press CAM AIR BUTTON, when lid opens take box out and place
new one in from film desiccator. Press CAM AIR BUTTON again so that vacuum pumps will
turn on. Close nitrogen gas valve in the adjacent room.
DO NOT walk away from the microscope until VACUUM STATUS goes back to READY, which
CM 200 Instructions 10
should happen in about 5 minutes.
4) Reset camera film counter
Press TEM CAMERA, then CAM INTT, then RESET. Hit RESET, it will now show 55
pictures (the stock of a new cassette).
SHUTING DOWN MICROSCOPE AT THE END OF THE SESSION
Follow steps 1-4 above as required, then:
5) EMPTY cold trap dewar
By pouring liquid nitrogen on the floor and let it dry, place paper towel in place of the dewar.
5) REMOVE cryostage
Pull sample holder out as far as it will go, then rotate it clockwise to 5 o'clock position (keep
Styrofoam container underneath because liquid nitrogen will pour out), continue to carefully
extract the holder from the airlock.
Cover holder tip with protective tube and connect cryostage to the heater. Set heater
selector to DEWAR (yellow light should come on), and let the cryostage warm up unitl the
temperature reads ~ 60C. Take the cryostage to the pumping station, and open vacuum for
the tip only to dry it out.
7) WRITE final P3 value, etc. into logbook
CM 200 Instructions 11
FILM DEVELOPING (in the dark room)
1) Sign logbook, check that there have been no more than 250 images developed including
yours, otherwise change DEVELOPER (see instructions below).
Check FIXER with a few drops of Hypofix solution. If white cloudy trace does not disappear the
fixer is not good and you need to change it (see instructions below).
2) In the dark (only red lights are allowed) put film into the special containers.
3) Put racks with film into DEVELOPER for 9-12 minutes. Actual time depends on whether your
images were under or over exposed. For underexposed images use 12 minutes, for
overexposed use 9 minutes.
4) While waiting for developer put new film into the cassette, when putting the stack remember
that film side should be up, also remember to check the position of the cut corner (it should be
top right).
5) Wash in WATER for 30 sec to 1 minute.
6) Put containers into FIXER for 4-5 minutes.
7) Put in WATER for 20-30 minutes.
8) FOTOFLOW rinse, shake off excess.
9) Push film up a bit on the rack and put in drying oven for 60 minutes.
10) Put cassette (next cassette and boxes with negatives) into desiccator and pump for 30
minutes.
CHANGING DEVELOPER
We use Developer D19 (full strength). Use one pack per 3.8 liters (one bath). Mix and stir for 10
minutes.
CHANGING FIXER.
New fixer is located in the cabinets across the tab. Read instructions on the bottle. One bottle
should be good for one bath (3.8 liters).
Developer and Fixer WASTE are disposed of in the same large container.
CM 200 Instructions 12
TROUBLESHOOTING
Problem
The extraction limit is set too high/low. How do I get to the right value?
Solution
Click so that the extraction limit is not lit; rotate the filament knob clockwise until you reach
desired value of extraction limit. Click the extraction limit again it will become lit and now it will
be the same value as you dialed. (If you want to decrease the value you dialed rotate the
filament knob counter clockwise)
Problem
Image moves.
Solution
Tap lightly the sample holder and/or touch joystick X/Y slightly.
Problem
Message is flashing on the screen.
Solution
Erase by pressing RESET button.
Problem
Focus seems to be way off. Auto-focus does not work. How do I focus?
Solution
First check the eucentric height. Once it is set properly, AUTO FOCUS should work. If it
doesn’t, go to the lower MAG. Find some dirt on the specimen and look at the Fresnel fringes,
when they start to disappear it means that you are close to focus. Find the position of minimal
Fresnel fringes, this should be close to focus. Go back to high magnification and focus as
usual.
Problem
Increasing or decreasing the magnification produces an appreciable movement of the beam or
the image.
Solution
Column Alignment is required.
Problem
A shift of the beam is accompanied by a tilt, or vice versa.
Solution
Do Pivot Point Alignment.
Problem
Joystick doesn’t move right.
Solution
Check that JOYSTICK POWER is on 2.00 and SEPARATELY should be off for simultaneous
XY movement.