These are instructions for use of the JEOL 4000FX IVEM and AEI EM-7 MkII HVEM at Albany. At the present time, the Teitz system cannot be used to record low-dose images on film with the IVEM.

The following sections are included:

IVEM

Labelled picture of the IVEM

Detailed description of the IVEM

HVEM

Updated 5/28/98, M. Marko

IVEM

Use of the JEOL built-in low-dose kit (MDP)

General notes

1. Check the film sensitivity setting. For SO-163 film developed for 12 min. in full-strength D-19, use sensitivity 15 for 200kV or 13 for 400kV. If a different value is shown on CRT page 1, type "sens nn".
2. In setting up the illumination conditions, note that at 200 kV, the condenser must be set by turning ANTICLOCKWISE from crossover. At 400kV, the condenser is set CLOCKWISE from crossover. This avoids vignetting by the objective aperture and allows maximum image shift. All three setup positions must have the condenser adjusted in the same direction from crossover.
3. Three setup positions are provided: SEARCH, PHOTO, and FOCUS. After the initial setup is completed, the position is switched from SEARCH to FOCUS when the "return" key is pressed, and only goes to PHOTO when the photo button is pressed.
4. For initial setup, and for modification of the setup during a run, the setup position can be set directly by typing P,F, or S.
5. Any desired magnifcation, image shift, and illumination conditions can be set independently for each of the three setup positions, however we use the the following conditions:

Setup procedure

1. Type MDP. The PHOTO position will be selected at first.
2. Set the magnification to 5000X and put a feature in the middle of the viewing screen. Adjust the illunination conditions and the position of the objective aperture. Make sure the image shifts are set to zero (both lights above the knobs should be on).
3. Select SEARCH and FOCUS positions, and duplicate the same conditions in all three positions. (This procedure is advised so all positions will have the same starting point.)
4. Select SEARCH. The magnification should still be 5000X, and the feature should still be in the center of the screen. Move the feature about 2cm to the left with the stage translations, then move it back to the center with the image shifts. (This procedure is necessary to accomodate both the video and the photo "detector positions" within the range of electronic image shift).
5. Select PHOTO and FOCUS, and center the feature with the image shifts (not stage translators) in both cases).
6. Select SEARCH. Move the feature to the video position (about 3cm to the left of the screen center) using the image shifts, and recenter the illumination. Set the illuminated area to about 1.5cm diameter, and set the spot size to as small as possible (hopefully 7) which gives a useable TV image. Lift the screen to see the TV image, and refine the centering of the feature and illumination on the TV monitor.
7. Select PHOTO.
8. Keeping the feature in the center with the image shifts (NOT the stage translations), increase the magnification as desired.
9. Recenter the illumination and set the size of the illuminated area to just fill the film area, or the area on the specimen you want to record.
10. Check the voltage center with the binoculars using the HT wobbler. Adjust using the beam tilt controls, if necessary, then readjust the illumination.
11. Check the centering of the objective aperture.
12. Set the exposure by adjusting the spotsize and bias controls (but don't exceed the permissible beam current value!!). Check the exposure with the small focussing screen to give a 1-2 second exposure.
13. Set the exposure mode to manual and select the closest available exposure time. NOTE: Make the exposure time a bit longer than required for the untilted image so that it will be adequate once the specimen is tilted to 60 degrees during tomography.
14. Select SEARCH, lift the screen, and check to see that the feature which was at the center in PHOTO is in the center of the TV monitor. If not, adjust the image shifts.
15. Select FOCUS. Set the magnification to the same used in PHOTO, and keep the feature at the center of the screen.
16. Move the feature about 3cm to the right with the image shifts.
17. Set the illumination to about 1.5cm diameter, and select spot size to 6 or 7 .
18. Move the illuminated area to the TV position (about 3cm to the left of center) with the illumination shifts.
19. Lift the screen and check centering of the illumination to fill the TV screen.
20. Check all positions again and touch-up any position which might have changed slightly.

Taking single low-dose images

1. Make sure the screen is up.
2. Select SEARCH and find an object to photograph.
3. Press return to get the FOCUS position.
4. Focus the image and press the photo button to take the picture.
5. NOTE: You will see a faint flash on the TV screen when the picture is being taken. If you see no flash, you will not have an exposed negative.
6. The SEARCH position will be automatically selected after photography, but you must raise the screen by pushing the button.
7. Look for the next object.
8. If moving to another part of the grid, the setups may have to be "touched up".
9. When finished, press the "ESC" key to stop MDP.
10. NOTES

• No commands other than MDP commands can be issued to the microscope computer while MDP is engaged.
• The setup is saved, so MDP can be restarted with the same setup after, for example, a film change (which may require a command to set the film stock number), or if the microscope has a problem and has to be restarted in some way.

Collecting a tilt series

1. Select SEARCH and locate the object to be recorded.
2. Tilt to -60 degrees while following the object.
3. At 60 degrees, refocus and check the PHOTO and FOCUS positions. The change of focus will likely require readjustment of the size position of the illuminated area. NOTE: It will occasionally be necessary to check the PHOTO area and reset the illumination and specimen position during the tilt series, if you notice that the focus is changing rapidly and by a large amount (i.e. more than 10 microns).
4. For cryo-tomography: set the TV sto manual so the thickness of the ice can be judged. Find a good area of ice thickness, with thicker ice (or carbon) to the left for focussing.
5. Don't forget to look for the flash on TV screen during exposure

HVEM

Low-dose and tomographic series recording on the HVEM

Under construction!

The HVEM does not have off-axis focus or tracking, however the TV-rate CCD camera on the HVEM has more sensitivity and resolution than the IVEM's TV-rate camera. Thus, low-dose recording can be done with on-axis focussing and tracking, together with recording on sensitive x-ray mammography film. Great care must be taken in use of the sensitive TV-rate camera to avoid damage to the camera or scintillator. Procedures will be written for this and posted here.