Using the MBI Hitachi H-7000 Electron Microscope
Morgan / morgan@mbi.ucla.edu Notes v0.02
Preparing grids
Glow discharge (purpose: so our sample sticks to the grid).
Put grids shiny-side up on Whatman paper in a Petri dish.
Place Petri dish under the bell jar on the alumnium plate in the EM room.
Fully tighten bell jar valve, then loosen slightly.
Flip the switch at the rear left of the Maxima air pump beneath the table.
Slowly re-tighten valve on bell jar.
Leave for ~1min.
Plug in Tesla coil and turn off lights.
Turn off lights and discharge for ~10seconds, attempting to make discharge even.
Unplug Tesla coil and turn on lights. Turn off pump.
Put thumb on the end of the tube at the base of the valve. Loosen valve until it sucks on your thumb, then use your thumb to slowly let air in.
Grids (depending on weather) will be for for 15mins - 2hours.
Applying sample and staining glow-discharged grids
Ensure buffer is OK: phosphate is incombatible, for example. Ammonium acetate and Tris are OK.
Clamp grid by the very edge using tweezers, shiny-side up.
Apply sample to grid; a bulbous blob (~7ul).
Leave for ~60seconds.
Wick off with filter paper from the very edge and simultaneously add a blob of uranyl acetate stain (CARE! Toxic!).
Quickly wick off and add more stain twice more. We're rinsing with stain here.
Finally add a final blob of stain and leave for ~45seconds.
Wick off stain and dispose of filter paper in uranyl acetate waste bucket.
Push grid off into Petri dish by using a wedge of filter paper between the arms of the tweezers and gently pushing it off.
Leave grid to dry for ~5minutes.
Turning on the EM
Fill the dewars with liquid nitrogen
Rear (vacuum pump trap): use metal funnel. Twist sheath so as to allow for an air-hole. Use pencil to gauge depth of liquid nitrogen. Two or three litres is good. A full tank will last all afternoon. Twist sheath to close off hole when done.
Front (cold finger): Using thermal gloves, remove by twisting and lowering carefully so as not to break. Full to within a couple of centimetres of full. Replace as removed. Replace every 30 – 45 minutes.
Turn on scope: on lower right, turn key from Evac on to Col on.
Check vacuum meter just to the left of the key, for different chambers: should be 'all the way to the left'. Penning gauge is for high vacuum; Pirani for lower vacuum. Only the column using the Penning gauge; all others use Pirani. Both gauges have Pascals as units, however. Column should be approaching 0.01 Pa, gun is also important, should be smaller than 2 Pa. Note that these two leave the needle in approximately the same place on the dial!
Change date on Film number on CRT using the arrows, numbers and full stop buttons on the keypad to the left. Enter key is the worn key with no visible lettering.
Preparing to view
Pull side lever up to reflect electrons into the binoculars. Be aware that spheres will now look like ovals!
Move binoculars over to the centre of the screen.
Adjust appropriately using wheel to adjust left eye focus, right eyepiece to adjust right eye and move the two eyepieces nearer or further apart to adjust for eye distance.
Press Ready/off on keypad on left once. From here, to turn off, hit button once more.
Select potential difference: go for 75kEv. To turn off now, need to press Ready/off twice.
Flip Panel lamp on.
Turn off the room lights and let the machine and your eyes warm up!
Preparing beam and image
Set beam to 75kEv.
Pull objective aperture out by turning the switch (just below and to the left of the sample chamber) to the right.
Turn up Filament knob until it registers on the voltage/current metre, about 75% of maximum output.
Set resolution to about 10 000x.
Use Brightness knob (which focuses the electron beam through the condenser aperture) to adjust the spot size to crossover. The beam will have an image on it, that of the 'filament' image itself.
Increase the Filament output until the spot is uniform in intensity (i.e., image is no longer visible).
Use Brightness centering knobs to move the spot to the center of the screen. Use the condenser apperture to make sure either side of crossover is centered on the same spot, i.e., that it's symmetrical.
Use diffraction mode to align optical axes and fully focus beam.
Go into diffraction mode by hitting Diff.
Make the diffraction spot v. sharp using the Diffraction spot knob.
Move the two translation knobs to center the spot.
Move out of diffraction mode by hitting the Zoom button.
Push objective aperture back in for high contrast (we will always do this).
Focussing
First, at ~10k magnification put the Holey grid in (grid sample #1, see below for loading grids).
Shift brightness to get a small, brighter spot and use focus to get a rough focus. The lower knob is coarser, the upper knob if fine. Also there's an additional Fine/Coarse switch.
Now shift to high magnification (~60k)
If there's a bright fringe inside the object, it's over focus. A bright fringe outside the object is under focus. Be a little under focus (3-4 clicks anticlockwise of the coarse knob in fine mode) if possible as this accentuates object boundaries.
Focus using the Focus dial.
Use the F/C knob to toggle coarse focussing mode.
Loading and retrieving grids
Placing grids in sample holders
Always use tweezers at a very shallow angle when dealing with grids. Never touch stuff with bare hands. Use optical lens paper for gripping stuff.
Loading sample holders
Ensure Ready/off is toggled to off and the Filament knob is rotated fully counterclockwise before loading or removing grids.
Check to ensure the specimem door is in Lock position.
Flick the switch from Evac to Air with finger holding sample chamber door shut. Should be able to hear door release.
Pull open specimen holder door.
Use tongs to grasp the back of a sample holder (don't use sample chamber 1 as it contains the holey grid), and remove and place on lens paper.
Remove thin metal sleeve from sample holder, put it on it's end with the rim at the bottom.
Use tweezers to hold the grid, ensure the shiny side is facing downwards, and slide into the thin metal sleeve. Once it's in, ensure it's not wedged at an angle at the base of the sleeve.
Insert grid holder into sample holder using tongs and remove to the side as they can fall out extremely easily. Note, that if there's a choice between dropping the grid holder and letting it fall to the floor, choose to catch it!
Hold sample holder door closed with finger and switch Air to Evac.
Pump a couple of times further by rapidly switching back to Air and returning to Evac.
Now, hold sample door closed with fingers and rotate knob as far as it can go opposite the arrow direction, and pull it towards you. It'll come about a couple of centimetres. This enters the sample holder chamber to the column, resulting in vacuum.
Turn now to the sample holder you require and proceed with Loading.
Loading grids into column
Note that there's are not safeguards to prevent multiple grids being deposited on top of each other... be careful...
Ensure (x,y) = (0,0) as displayed on CRT.
Ensure no grid already in the scope.
Align line on sample chamber window to desired grid holder.
Rotate from Up to Pick up then back to Up. Pick up picks up the grid. Set would deposit it.
Inject grid into column by flicking switch at top right of the sample chamber Out to In.
Now rotate from U. to S. then back to U.
Pull sample injector out by flipping switch on top right of sample holder back from In to Out.
Leave it with Lock in.
Retrieving
Ensure that when picking a grid from inside the scope to ensure that (x,y) = (0.0) using the translate knobs.
Inject (empty!) sample injector into scope.
Switch from U. to P. back to U.
Flip switch to withdraw injector from scope.
Move line on sample chamber window to empty space in sample holder.
Switch from Up to Set back to Up.
Talking to the EM computer
To go to one of the six specific menu screens, press the corresponding number, then hit Index.
On the Index screen [???] there are different options...
Taking photos
Be quick, as the beam damages the grid!
Use Index to toggle to screen with magnification on it. Note film number and portion of negative exposed. Make a note of it!
Adjust Brightness to make sure we have a green LED for Density adj. Note that if we have a large amount of dark in the image that the overall density reported by Density adj will be an average. Take it into account. Thus in this example, have a little lower than a green LED.
Be a little under-focus (see focussing). At 30 000x, ~3 clocks anticlockwise on the bottom focus knob (in fine mode) works fine.
Tilt phosphorous viewscreen all the way back.
Pull screen tilt lever all the way towards you and hold with the lights all off.
Note: never leave a film with just one half exposed!
Note this in the photo log.
Loading film
When there are only ~5 unexposed films left, let Mari or Martin know.
Fingerprints matter!
Use red 'safe' light if you need a light. Don't shine it directly on the film though!
Box can accept 22 negatives. Each negative takes two images.
Be sure to place the cover plate on top; the top plate is always rejected by the machine.
There is a second box for taking exposed film.
Retrieving films
Turn on the stage lights
Get empty replacement receiver boxes
The camera is directly under the column and has three buttons: Evac, Close and Air.
Press Air to let air in and leave for approx. 1 minute to equilibrate to atmospheric pressure.
Turn off all lights.
Open camera door
Feel around and pull out film box and replace with empty box.
Close camera door
Hold the door and press Evac camera button. Hold the door closed until you feel it go.
With safe light, remove the films from their holders and develop immediately or put on storage box.
Developing films
Fill in log
Run water with thermometer in flow. Adjust to 20 degrees.
Use plug/overflow combo plastic tube to allow semi-filling of basin. Fill.
Leave developing containers to equilibrate for 30mins. Developing the film is directly dependent on both time in development and temperature of developing solution.
Come back after 30 minutes and line up the box with undeveloped film and film rack so as to be close-to-hand.
Put 'Developing film' note on door, close door, draw curtain, turn on 'Developing film' light, turn on safe light (red), turn off all other lights.
Open film box on very edge of safe light illumination. Carefully slot undeveloped film into film rack. It helps here to use the lid of the film box as a shroud to protect the film still in the box.
Set timer to 3:45. This is crucial.
Put rack into developing solution. Leave outside lid on the side; inside lid floating in water. Start timer.
Meanwhile, fill bucket with water.
After time is up, move rack into water for 30s. Close developer solution box and ready fixer.
Move rack to fixer solution for 10 minutes.
Meanwhile, replace water in bucket.
Again, rinse rack with water for 30s.
Move rack to Hypoclear for 2 mins.
Meanwhile, fill bucket with distilled water.
Move rack to bucket and leave for 5 minutes with water running slowly.
Move rack to dry overnight on paper towels. Empty out bucket and leave upside down. Drain basin.
Turning off the EM
Adjust the Brightness knob to diffuse the beam.
Turn down Magnification to ~10 000x.
Turn down Filament.
Hit Ready/off twice.
Make sure you retrieve all sample grids and replace all grid holders.
When in Lock position of the sample holder, turn the sample holder knob in the direction of the arrow, then push knob in. The red light near the Air/Evac switch should come on momentarily, then go off.
Switch sample holder to 'lock' and push in sample wheel.
Turn key from Col on to Evac on. Don't turn further as it'll shut down everything, including the vacuum pumps.
Fill in logbook, including photos taken in the photo log.
Put viewscreen cover on.
At end of session, remove exposed film box and either develop immediately or put in light-tight boxes. Ensure you remove the cover plate from the bottom, too.