This document contains detailed instructions on how operate the Tietz computer system and CCD camera on the IVEM. It only applies to the installation at Albany.

Labelled picture of the IVEM

Detailed description of the IVEM

updated 5/28/98, M. Marko

This document includes the following sections:

Detailed setup and use of automated tomography

• Initial setup
• Adjust goniometer stage
• Adjust aperture
• Calibrate autofocus
• Calibrate tomography
• Initial setup for automated tomography
• Setup for start of tomography
• Setup of tomography conditions
• Setup tables
• Starting tomography

Other procedures and applications

• Initial goniometer adjustment for best double-tilt tomography
• Use of the TV camera without the Tietz system
• Minimum automation mode
• Sample setup for cryo-tomography

Initial setup

1. Check that the time is set to 1000ms.
2. Select FORMAT.
3. Turn on beam blank.
4. Select PHOTO to turn off beam blanker and lower screen.
5. Set exposure to about 40pA on the small focussing screen if test CCD exposures are to be made before proceeding.
6. Be sure the microscope is aligned well for the conditions to be used. Note the special alignment of the condenser system for minimum movement of the illuminated area with focus change.

Adjust goniometer stage

(See below for initial set-up of the goniometer, and for specimen insertion instructions, to yield the maximum open area at high tilt, and to allow the possibility of stage alignment under low-dose conditions. ADJUSTMENT OF THE STAGE ALIGNMENT SCREWS IS ONLY TO BE DONE BY IVEM STAFF.)

1. Set the mag. to 6000X. For 400kV only, set the condenser minilens (CM) to PRTEST M1: 66BA using the command SETLNS 3 &H66BA typed on the EM keyboard. This command is defined as the "F4" macro--press F$, then return.
2. Take up the rotation backlash for a double-tilt series: Drive the rotation of the stage using the computer to -20 degrees, then back to zero. Later, for the second tilt, you will rotate the grid in the positive direction (using medium speed) to 90 degrees. NOTES: (1) If you need to pre-orient the specimen by rotation before the first tilt series, you will still need to take up the backlash after you determine the starting rotation position. (2) If using the foot pedal or pushbutton switch for rotation after the first tilt series, be sure the switch is set in the positive direction.
3. Adjust the stage height (z) for minimum movement of the specimen in the range +/-30 degrees.
4. Put a feature at the center of the screen at zero degrees tilt using the stage translations.
5. Tilt to -60 degrees.
6. Turn the screw to the left of the objective aperture (using a 5mm hex key) to move the image back to the center. Note that the image may move off to one side, as well as up or down (i.e. it may move in both x and y at the same time). Ignore the movement to the side (x), just put the image on the same vertical (y) level as the center of the screen.
7. Tilt back to zero degrees and recenter the object with the stage translations.
8. Carefully readjust the stage height for minimum movement, while tilting through the whole range (a very small change in height can make a significant difference in movement).
9. Repeat the alignment if the image moves more than 2 cm when tilting from -60 to +60. Try to keep the movement not more than 1 cm.

Adjust aperture

1. Set the specimen to the eucentric height and focus the image, if not already done.
2. Set the image shift to zero.
3. Make sure the CM is set to 66BA (macro key F4) if using 400kV.
4. Set the spotsize and illumination to the conditions to be used for the FOCUS position of the tomography program.
5. Center the aperture.
6. Check the intrusion of the aperture using the x image wobbler at ampltude 2, frequency 1. The center 1 cm of the field should remain clear.
7. NOTE: The aperture centering will have to be redone during the tomography-series setup, if the focus or position of the focus area is changed significantly.

Calibrate Autofocus

(This may not be needed if previously done at the selected magnification and CCD format, but the previous correction might not be ideal for the current specimen.)

A. For 5000X or 6000X:

1. Be sure the CM is adjusted according to the instructions above (66BA for 400kV; default if 200kV), and the focus, spot size, and illumination size are set as they will be during the tomography series.
2. Be sure the image is close to focus.
3. Set the exposure to about 40pA/cm**2.
4. Set the time to 1000ms.
5. Set the format to 512x512, center, 1X binning.
6. Set target to 0.
7. Set offset to 0.
8. Set beam tilt to 1.0 for aperture 3. (less for aperture 4)
9. Set focus incr. to -20000 for aperture 3. (less for aperture 4)
10. Select CALIBRATE.
11. If the aperture appears during the calibration, check the conditions set up in step 1.
12. If OK, save the calibration data.
13. Go to PHOTO mode and check the intrusion of the objective aperture using the x image wobbler at ampltude 2, frequency 1.
14. Try the autofocus correction (target -300, offset 2000) and make sure the aperture does not appear.

B. For 25,000X, use beam tilt 1.0, focus incr. -2000.

C. For 10,000X use beam tilt 3.0, focus incr. -10,000

Calibrate tomography

(Not needed if the previous series was done with the same parameters, the same specimen rod is used, the specimen is centered on the grid, and the microscope aligment was not changed).

1. Be sure the condenser mini-lens is set to 66BA, if 400kV is used, and the focus, spot size, and illumination size are set as they will be during the tomography series.
2. Set the camera format to 512x512, full size, 2X binning.
3. Set the exposure on the small screen to about 20pA/cm**2, even if it means the size of the illuminated area needs to be increased.
4. Set the camera rotation as follows , or use alternate method, step 5.
   1. Make sure the specimen is at the eucentric height. (400kV: Objective voltage approximately 7.445V) (200kV: Objective voltage approximately 6.41V)
   2. Select the magnification to be used.
   3. Rotate the grid until a grid bar or other straight line is along the x translation direction.
   4. Rotate the camera until the line is exactly horizontal on the CCD. Note: 5000x and 6000x have the same rotation.
5. Set camera rotation, alternate method.
   1. Make sure the specimen is at the eucentric height. (400kV: Objective voltage approximately 7.445V) (200kV: Objective voltage approximately 6.41V).
   2. Select the magnification to be used.
   3. Move a gold marker on the specimen with the x translate knob only, and take a CCD image at two different x positions. Sequence the images so that the the two images are side-by-side on the monitor.
   4. Use a straightedge to check if both markers are the same distance from an x-edge of the image frame on the monitor: If the line between the two marker positions is not parallel to the x-axis of the image, rotate the camera a little and try again.
6. Select TOMOGRAPHY.
7. Select calibrations (Y). The "goniometer alignment" is not done here (already taken care of with camera rotation). The "real" goniometer alignment is described above.
8. Select E (Electron Optics).
9. Lift the screen if it is not already up (with button on EM console).
10. Select I (image shift calibration) to run calibration.
11. Select B (beam shift) and follow the prompts (screen is automatically lowered). Do this with a very small illuminated area.
12. Lower screen (with button on EM console).
13. Reset illumination size to that to be used for tomography.
14. Select F (calibrate condenser for focus change) and follow screen prompts.
15. It may be preferable to do this focus calibration at -60 degrees, since the calibration range will then match the correction range in direction of focus change. IMPORTANT: The illumination knob is turned CLOCKWISE to make the spot bigger at 400kV and COUNTERCLOCKWISE to make the spot bigger at 200kV
16. Save data according to prompt while exiting calibration.
17. Exit tomography and reset the camera format to 1024x1024.

Initial setup for automated tomography

1. Do the initial setup, and the stage and aperture adjustments, as above.
2. Be sure the camera is rotated to the correct position for the magnification to be used.
3. Be sure the tomography calibrations are already done for the conditions to be used.
4. Be sure autofocus is already calibrated at the magnification to be used, and set for 1000ms/512x512 1x binning, center.
5. Set autofocus to -300 target, 2000nm offset.
6. For 400kV, be sure the condenser mini-lens is set at the correct value (e.g. SETLNS 3 &H66BA)

Setup for start of tomography

1. With the stage at zero tilt, center the object of interest (or an object of similar density)
2. Make sure the backlash for the rotation has been taken up (see stage alignment, above).
3. Be sure the image shifts are at zero.
4. Adjust the illumination size according to the table below, and set the spot size and bias in combination so that the CCD exposure in the camera mode to be used for the tilt series is just below saturation (i.e. max less than 2900, mean about 2000). Lower values can be used for sensitive specimens, but some shot noise will appear.
5. If the spot size or magnification is changed, reset the condenser mini-lens (CM).
6. Switch back to Photo after checking exposure.
7. Drive the stage with the foot pedal, pushbutton, or joystick (use medium speed!) to the maximum negative tilt value (usually about -61 degrees), recentering the object of interest. You may increase the spotsize to make a brighter image for chasing the object, but be sure to return it afterwards (and reset the condenser mini-lens) if you change the spot size.
8. Set the tilt to -60 degrees using the stage computer keyboard and recenter the object.
9. Refocus using video and check exposure (should be less than at zero degrees).
10. Check autofocus aperture centering using the x image wobbler at amplitude 2.

Setup of tomography conditions

1. Select tomography.
2. Make sure the CM is at the correct value.
3. Lift the screen using the button on the EM console.
4. Check focus on the TV monitor using (V).
5. Return to photo position using (F) "beam blank". If the beam is not visibile, press (F) again.
6. Check the illuminated area size and centering.
7. Lift the screen using the button on the EM console.
8. Check exposure (E) (should be less than it was at 0 tilt).
9. Update the scanning position (U).
10. Return to photo position using (F) "beam blank". If the beam is not visibile, select (F) again.
11. Set directory and file name (B).
12. Copy (R) the current (scanning) position to all the other positions.
13. Go to search position (C,S) and do following steps.
    1. Set the magnification according to the table below.
    2. Set the spotsize one step smaller (e.g. from 6 to 7).
    3. Re-set the condenser mini-lens by typing on the EM console SETLNS 3 &Hnnnn, where nnnn is the previously-determined hex code value (66BA for 6kX, #3 obj. aperture. 66BA is defined for the macro key F4: Type F4 and return).
    4. Adjust the illumination size according to the table below.
    5. Accurately center the illumimation.
    6. Shift the image (L) according to the table.
    7. Check that the illumination is centered. If the illumination is not centered, the image shift and illumination shift calibrations must be redone, although slight mis-centering is OK.
    8. Check the camera setup and select (N) if necessary according to the table below.
    9. Check that the object of interest is not visible on the screen and that the illumination is centered (if not, try a different image shift value). Do not readjust the illumination shift.
14. Update the search data (U).
15. Go to focus position (C,F) and do following steps.
    1. Set the illuminated area according to the table.
    2. Accurately center the illumination.
    3. Set the image shift (L) according to the table.
    4. Check the camera setup (N) and selectthe proper format if necessary.
    5. Check the aperture position using the wobbler.
    6. Check that the autofocus parameters (M) are set properly. (Use 0 target, -3000 offset for 6kX).
    7. Check that the object of interest is not visible on the screen (if it is, try a different image shift value), and that the illumination is centered. Do not readjust the illumination shift. If the illumination is not centered, the image shift and illumination shift calibrations must be redone, although slight mis-centering is OK.
    8. Note that if you change the magnification or spotsize using the microscope controls, you will have to reset the condenser mini-lens.
16. Update the focus data (U).
17. Copy (R) the focus data to tracking.
18. Go to tracking position (C,T) and do the following steps.
    1. Set the magnification if it is to be different from focus (normally it is not).
    2. Increase the spotsize one unit (e.g from 6 to 7).
    3. Re-set the condenser mini-lens by typing on the EM console SETLNS 3 &Hnnnn, where nnnn is the previously-determined hex code value (66BA for 6kX, #3 obj. aperture; use marco key F4).
    4. Check the camera format setup (N) and re-set if necessary.
    5. Set the illumination size according to the table.
    6. Check that the illumination is centered, but do not adjust it if it is not. It should be the same as for the focus area.
    7. Update the tracking data (U).
19. Select the expose area (C,E).
20. Check that the object of interest is still centered.
21. Check the camera parameters.
22. Raise the viewing screen with the button on the microscope.
23. Select video (V) and refocus the object.
24. Make an exposure (E) and see that the object of interest is centered.
25. If not, select video (V), and recenter the object.
26. Check that the expoure values are correct (max about 2000, mean about 1000-1300 for 0.25 um stained sections). For thicker sections, see notes at the end of these instructions. Lower values will have noticeable shot noise.
27. Update the exposure data (U).
28. Go to each of the three other positions in turn (F, T, S) and check that the exposure is correct. This also records sample images at the right of the screen.
29. Set up the tilt series parameters (J). Use a two-degree interval for all four image shift positions. Do not use fully-automated tomography for now.

Setup-tables

Image shift for low-dose work

Exposure mag. X1000	Exposure shift, nm	Tracking/Focus shift, nm	Search mag. X1000	Search shift, nm
6	0	5000	-	-
15	0	2500	6	4000

Camera format

Exposure	1024x1024	full	no binning	1000ms
Focus	512x512	center	no binning	1000ms
Tracking	1024x1024	full	no binning	1000ms
Search	1024x1024	full	no binning	1000ms

Illuminated area size (screen scale divisions)

Exposure mag. X1000	Exposure	Focus	Tracking	Search
6	5	8	6	---
15	6	6	6

Starting tomography

1. Press S to start.
2. After each tilt, the program will ask you if the shift is correct. If it is not:
   1. Select V and center the object with the stage translation.
   2. Select E to see the acquired image.
   3. If OK, select Z and see the cross-correlation.
   4. If OK, select O for OK to continue. You will have to repeat this at the tracking step, as well.
3. Note the beam current value at the start of the series. As the series progresses, adjust the filament voltage to maintain the same beam current.

INITIAL GONIOMETER ADJUSTMENT for best double-tilt tomography

• Ensures largest visible area at high tilt, and avoids collision with objective aperture holder.
• Must be done each time the tilt-rotation holder is put back into use after another holder has been used.

Initial calibration:

1. Place a slot grid (with a specimen in the center of the slot) in the specimen holder. Be sure the support film side is up and there is an anit-twist washer both below and on top of the grid.
2. Insert the holder in the microscope and home (H on stage computer keyboard) the stage.
3. Select low magnification (e.g.100-400X)
4. Adjust the specimen selector knob to center the grid.
5. Rotate the grid through a large angular range to locate the rotation center.
6. Place an object at the expected rotation center by moving the x and y translations. The center shifts a bit during rotation, so this can't be done perfectly.
7. Reset the zero for the x and y movements on the stage computer.
8. Rotate the grid so that the edge of the slot is parallel to the tilt axis, after first taking up at least 20 degrees of backlash.
9. Reset the zero for rotation on the stage computer.
10. Go to 6000X, set the eucentric height, and align the goniometer for minimum specimen movement during tilting.
11. The objective voltage should be 7.445 at focus (for 400kV), at all magnifications.

To set up for each specimen change, after the initial calibration has been done:

1. Before removing the holder, be sure the rotation has been set at zero, after taking up the backlash.
2. If using a slot grid, keep the long axis of the slot grid parallel to the long axis of the stage rod.
3. Be sure the support film is facing up and there is an anti-twist washer both below and on top of the grid.
4. After insertion, home the stage and set the height to zero.
5. Select an exposure area as near the home location as possible.
6. Move to an adjacent area on the specimen, using x (right hand) translation only, and align the goniometer.
7. Move back to the exposure area.
8. An automated tilt series should now be possible, with a tilt increment of 2 degrees or less.

Use of the TV camera without the Tietz system:

1. Turn off switch on shutter interface unit.
2. Set F1 to DFCABS 11 &H0000 &H0003 (Corresponds to 7C18 66B0)
3. Set F2 to DFCABS 11 &HB230 &H59B3 (Corresponds to 2E48 C063) (FKEY 2 DFCABS 11 &HB230 &H59B3)
4. To move the image to video position, press F2, return, then lift the screen
5. To moce back to the photo position (screen center), press F1, return, then put down the screen.

Minimum automation mode

1. Use only exposure and tracking, both with the same parameters, including camera parameters.
2. Turn off autofocus and auto tracking. You might add search for initial location of the object or if tracking is expected to be difficult.
3. Select "D": manual tracking on.
4. Do coarse tracking and focus manually in video mode.
5. Check the exposure by looking at the last tracking image.
6. If the picture is not good, click on "no". The tracking position is recalled.
7. If the illumination shifts, use "update" in tracking (or search). The screen will go down, and you can reset the illumination. The changes made will be applied to all the other positions

To copy multiple files

copy -w=(directory) (filenames using *)

SETUP FOR CRYO-TOMOGRAPHY, 200kV, "15kX" (0.7nm pixel size)

Steps 1-26 can be done on the previous day, and don't need to be repeated if the microscope continues to be used for "15kX, 200kV cryo"

1. Make sure the image center is the same for 6kX and 15kX .
   1. Center a feature at 15kX.
   2. DADJ 1.
   3. Go to 6KX, recenter with projector align shift.
   4. Go to 15KX, recenter with image align shift.
   5. Repeat until centered for both mags.
   6. DADJ 0
   7. Rotate camera to correct position.
2. Align goniometer in center of grid with cryo holder, using test specimen.
3. Use beam current setting 4-6; 63.0-63.3 uA.
4. Start tomography menu.
5. Set (A)scanning to 6kX, image shift centered, spot 6.
6. Copy to other positions: S E F T 7. Set (A)scanning to 2x binning, spot
7. Update
8. Do I and B calibrations.
9. Go to (E)xposure 1x bining, 15kX, image shift centered, spot 6. BE SURE illuminated area is about 2cm diameter.
10. Copy (E)xposure to (F)ocus and (T)racking.
11. Center feature on CCD in E, check intensity.
12. Go to (A)scanning, and check mark on video monitor to see that the feature centered in (E)xposure on the CCD is on the mark on the video monitor in (A)scanning.
13. Go to (F)ocus.
14. Do calibrations I and B.
15. Set L=1250 (halfway between (F)ocus and (E)xposure positions).
16. Check condenser alignment for objective defocus shift:
    1. Turn on C alignment.
    2. Focus image, reset focus readout.
    3. Set focus to -20um, resize illuminated area.
    4. Center with C DEF.
    5. Set focus to +20um, resize illumination.
    6. Center with C SHIFT.
    7. Repeat if neeeded so illuminated area (when made the same size in both cases) is centered at both defocus values.
    8. Turn off alignment.
17. Set autofocus to +2000 offset, -300 target for in-focus (add desired underfocus to -300)
18. Set L=-1250, check calibrations by noting if illumination stays centered.
19. Set L=2500.
20. Copy (F)ocus to (T)racking.
21. Go to (S)earch, set L=4000 (make sure centering is maintained, checking calibration).
22. Update (S)earch.
23. Set up file parameters (B) and series parameters (J). Use semi-automatic at first, at least.
24. Tilt to -60 (-61, then -60), refocus in (E)xpose.
25. Go to (F)ocus, calibrate focus change (size only, not position) and center aperture with wobbler.
26. Return to zero tilt.

To start a cryo series after above setup

1. Exchange test specimen for cryo specimen, center grid, check goniometer alignment in center grid square.
2. Tilt to -60
3. Move not more than +/- 2 grid squares, in X ONLY for searching.
4. Go to (A)scanning, center prospective object to reconstruct on mark on TV, focus and take CCD image for more clarity
5. If object might be OK, go to (E)xposure and take CCD image (E) to confirm, also to check intensity.
6. If object is OK, take CCD images in (S)earch, (Tracking) and (Focus) to be sure there is something to track and focus on, and that the intensities are OK.
7. If OK, start the series.

NOTE: If the tilt increment is to be 4 degrees, search still needs to be set at 2 degrees to properly follow the specimen

Recentering the oject in the image frame:

1. Must be in the semi-automatic mode.
2. At (T)racking, use C to move the cursor IN THE DIRECTION OF THE ERROR (e.g., if the object is too high, move the cursor up beyond the red circle)
3. When finished moving, use q.
4. Use S to apply the change 5. Use E to make a new reference image. The new reference image will have moved in the OPPOSITE direction as the cursor, i.e., in the direction you wanted to move the object.