User Instructions for 2100F

• Assumed that the microscope has already been aligned and software set up for imaging
• All users REQUIRE training with staff before using K2 camera
• In general, this microscope will be used only for cryo samples

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line

• Mag 1 button to reset Defocus

iMDS

New Instructions for K2 camera

• Please read and be familiar with use of Leginon and K2 on EMG website
• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• Dose (brightness) on all modes should be 10 pA/cm2
• 10.4 pA/cm2 = 8 e/pixel/second
• IMPORTANT: Beam MUST be blanked (live mode off) during mode changes, otherwise camera may be hit by bright beam
• Can run Leginon "Manual" app for movie collection
  • Set Camera Mode to Super resolution
  • Choose to record movies (frame numbers are irrelevant for K2; all are saved)
  • Choose total exposure time and frame exposure time as apprporate
  • Be sure that option to save images is checked (under Advanced Settings)
• Makes files smaller (raw images saved as bytes, gain correction and alignment done via Appion)

Good settings

• Search Mode
  • alpha 3 (75C0)
  • 2000X mag
  • To make beam dim enough, change 'Custom" to on and spot size FFFF
• Focus
  • alpha 2 (95D0)
  • 80,000X mag
  • Spot 5
  • Beam condensed to just cover camera (only viewing center 1/2 or 1/4)
• Photoset
  • alpha 2 (95D0)
  • 25,000X mag (~1.4 A/pixel)
  • Spot 3-5 (beam very condensed to just cover camera)

• Use DM for live search and exposure
  • Search: Full CCD, 4X binning
  • Focus: Center 1/2 or 1/4, 1X binning
• Use Leginon for exposure
  • images to database
  • 3/13/15 -- still saved on local disk, need to be manually transferred to bnc14

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with live view in DM (K2 camera) -- Use Search setting
• Find and center area of interest (use PLA to align Search and Photoset)
• Stop live viewing
• Go to Focus mode, change DM preset to Focus
• Put specimen in focus (zero contrast, Thon rings maximized)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Stop live viewing
• Go to MDS Photo Mode
• In Leginon software, press camera icon to take and save image
• Go back to Search mode. Change DM setting to Search

Advice and Common Issues

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In JEOL Oprate window, tilt up in 5 degree increments
• If you are not eucentric, you will see image shift on the live camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Reset tilt angle to 0 using JEOL interface. Should see no (minimal) sample movement

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
• If beam disappears when moving focus position, call a staff member to redo beam shift / image shift alignment

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• go to Search then Focus
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

Use STD Photoset to getr low-mag views of grid

• Go to Standard Photoset mode (normally not used at all in cycle)

1. Press Low Mag button and lower mag to 150X or lower
2. manually find and center good squares.
3. Store positions in JEOL stage interface for later recall
4. Go back to MDS Photoset when done. Leave for 15 minutes to allow lenses to warm back up

SerialEM

• 3/15/15 -- not yet installed on K2
• Never try to use SerialEM and MDS/iMDS at the same time
• Never use SerialEM with the standard tilt holder

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....

• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level

• Go to MDS Photo Mode

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest

• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• go to Search then Focus
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

SerialEM

• Never use SerialEM with the standard tilt holder

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximized)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get a flash on Fast scan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fast scan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execute tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot Trash.findDFdf Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fast scan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• go to Search then Focus
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this
• Do not worry about astigmatism in focus mode. Because of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Press Low Mag button and lower mag to 150X
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Raise screen and put CCD/TEM toggle in CCD mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window
10. Choose to make a full grid montage. Change overlap to 15% and choose to ignore pieces to skip.
11. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
12. Once done, choose to make a new map.
13. If map doesn't look good, try turning off or on "sloppy montage" option and see which is better. Double click map to reload
14. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag
15. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
16. Optionally, save navigator file
17. Quit SerialEM, go back to mag 1 mode
18. Cycle through hysteresis cycle a few times. You will likely need to realign things.
19. Restart EM Menu 3
20. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximized)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get a flash on Fast scan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fast scan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execute tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot Trash.findDFdf Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fast scan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• go to Search then Focus
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this
• Do not worry about astigmatism in focus mode. Because of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)

1. Center beam with beam shift, adjust brightness and spot size to 5
2. Raise screen and put CCD/TEM toggle in CCD mode
3. Quit EM Menu 3 if it is running
4. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
5. Set up Record at binning 2, and 0.1 second exposure
6. Take a test record image to be sure exposure is good
7. Open a new Navigator window
8. Choose to make a full grid montage. Change overlap to 15% and choose to ignore pieces to skip.
9. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
10. Once done, choose to make a new map.
11. If map doesn't look good, try turning off or on "sloppy montage" option and see which is better. Double click map to reload
12. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag
13. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
14. Optionally, save navigator file
15. Quit SerialEM, go back to mag 1 mode
16. Cycle through hysteresis cycle a few times. You will likely need to realign things.
17. Restart EM Menu 3
18. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera

• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image

• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute

• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)

• Close "control TEM" window

Cannot Trash.findDFdf Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values

• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5

1. Quit EM Menu 3 if it is running
2. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
3. Set up Record at binning 2, and 0.1 second exposure
4. Take a test record image to be sure exposure is good
5. Open a new Navigator window

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get a flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot Trash.findDFdf Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to Trash.findDFdf object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Do not worry about astigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get a flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times

1. Go to Focus, then MDS photoset
2. Lower screen
3. Use specimen movement to center the object manually
4. Go back to Search mode
5. Be sure that PLA button is lit
6. Use DEF/Stig X and Y to re-center object

Astigmatism in Focus mode

• Do not worry about astigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get a flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image

• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Stigmatism in Focus mode

• Do not worry about stigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Taking a film image

• To take a film image, be sure that auto-exposure is unchecked
• Also be sure that CCD/TEM toggle is in TEM mode, otherwise you will get a blank film image
• After adjusting focus in Focus mode, lower screen
• Click Photo button in iMDS -- should hear film being inserted, screen raised, and exposure light should flash
• After film image taken, you are brought back to focus mode
• Optionally, can take a CCD image after a film image to verify that region is ok.

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Stigmatism in Focus mode

• Do not worry about stigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

Stigmatism in Focus mode

• Do not worry about stigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha lapha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• Do not worry about stigmatism in focus mode. Becasue of beam shift and inaccuracy of centering beam over camera, you cannot rely on focus to set stigmatism
• If it is really bad, try recentering Focus using PLA as described above
• Instead, do this in Photoset mode at 50,000X mag using live Focus mode in DM
• When happy, write down Objective Stig lens values (in JEOL menu)
• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha lapha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging
• Some familiarity assumed, as these instructions are not nearly enough for a first-time user

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha lapha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha lapha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

• Note tha lapha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. Open a new Navigator window

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated

1. Once things are set up, press "start" under Montage window. Takes 15-20 minutes to complete
2. Once done, choose to make a new map.

1. Mark off good grid squares by using the "add points" function. Click the middle of the square to be sure you will hit it at higher mag

• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder

1. Quit SerialEM, go back to mag 1 mode
2. Cycle through hysteresis cycle a few times. You will likely need to realign things.
3. Restart EM Menu 3
4. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• Stop live mode, adjust brightness and mag to original values
• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good

1. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• In Focus mode, adjust Stig lens values to numbers written above (Photoset gets these from Focus, not vice-versa)
• SerialEM can be used for montaging while in iMDS as follows:

Apertures visible in Focus and Search

• With small condenser aperture needed for K2 , you often see the edge of the objective aperture in Focus and Search modes
• Can adjust bright tilt to make beam fully visible
• Note that each mode has its own bright tilt, so this will not affect exposure mode
• good idea to do a few hsyteresis cycles after changing this

1. Start SerialEM. Should hear a beep. If you get an error, quit and restart.

• If it is really bad, try re-centering Focus using PLA as described above
• At a 2 um radius, you often see the edge of the objective aperture in Focus mode

Use of SerialEM to montage Entire grid

• In general, should not use SerialEM and iMDS at the same time unless you know what you are doing
• it is particularly important not to adjust magnification or iMDS mode if both programs are running
• SerialEM can be used for montaging while in iMDS as follows:

1. Go to Standard Photoset mode (normally not used at all in cycle)
2. Go to each point by highlighting it and picking "Go to xy" in SerialEM. When there, save stage position in JEOL stage menu.
3. Center beam with beam shift, adjust brightness and spot size to 5
4. Quit SerialEM, go back to mag 1 mode
5. Quit EM Menu 3 if it is running
6. Start SerialEM. Should hear a beep. If you get an error, quit and restart.
7. Set up Record at binning 2, and 0.1 second exposure
8. Take a test record image to be sure exposure is good
9. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

1. Take a test record image to be sure exposure is good
2. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007

User Instructions for 2100F

• These are for users who come to use the microscope
• Assumed that the microscope has already been aligned and software set up for imaging

Controls needed

• F1 button -- lift screen
• Specimen movement trackball
• Focus knobs
• Brightness control
• Def/Stig in PLA adjust, in case beam gets out of line
• Z height to check eucentricity
• Mag 1 button to reset Defocus
• TEM/CCD toggle to change shuttering control to CCD

iMDS

Sequence of events

• Need to set up a "hysteresis cycle" of Search --> Focus --> MDS Photo --> Search ....
• In Search mode, lift screen and view display with Fastscan camera
• Find area of interest and center on Fastscan camera
• Go to Focus mode, and put specimen in focus (zero contrast, Thon rings maximised)
• Press Mag 1 to reset defocus to 0
• Adjust defocus to desired level
• Toggle TEM/CCD to CCD. Should see beam blank. Call for help if beam does not blank.
• Go to MDS Photo Mode
• In EM Menu 3 software, advance image number to a fresh image
• In EM Menu 3, press "Exposure". Should get s flash on Fastscan camera, and a new image should come up
• Optionally, lower screen for a few seconds to leave a good burn hole. Re-raise screen.
• Go back to Search mode. Change TEM/CCD toggle back to TEM to unblank beam

Problems

Eucentricity

• After you move very far (several grid squares), it is a good idea to check eucentricity, as sample may be bent
• In Search mode, center an object of interest
• In EM Menu 3 software, press Control TEM to open up a new window
• In Control TEM window, set X tilt angle to 15 degrees, and press execute
• If you are not eucentric, you will see image shift on the Fastscan camera window
• Adjust Z height until object of interest is re-centered (it will never be exactly recentered)
• Set tilt angle to 0, and execult tilt. Should see no (minimal) sample movement
• Close "control TEM" window

Cannot find Focus beam

• Focus beam may disappear if you have to adjust defocus a lot (indicating change in specimen height) or if you leave hysteresis cycle
• Bring it back as follows:
  1. lower screen to see beam
  2. Make sure PLA button is lit
  3. Move beam to center of screen using Def/Stig X and Y knobs (NOT beam shift knobs!)
  4. In EM Menu 3 software, click "Off-axis" button to move beam over Fastscan camera

Search and MDS Photo no longer aligned

• Search and Photo may go out of alignment, especially if you leave the hysteresis cycle

1. Before realigning anything, cycle through Search --> Focus -- > MDS Photoset a few times
2. In Search mode, go to a high-contrast and easy to find object, like a hunk of ice
3. Go to Focus, then MDS photoset
4. Lower screen
5. Use specimen movement to center the object manually
6. Go back to Search mode
7. Be sure that PLA button is lit
8. Use DEF/Stig X and Y to re-center object

SerialEM

1. Take a test record image to be sure exposure is good
2. In Search mode, go to saved positions and search there

SerialEM

• Be sure that you are using alpha setting 3, as that is where SerialEM has been calibrated
• TEM/CCD toggle should be in CCD position
• If beam seems to go crazy in low-dose mode, check that alpha setting is 3 in all imaging modes
• Note the alpha setting is not copied when you copy one mode to another: you must manually adjust it then choose "continuous update of mag and beam"
• For montages, use at least binning 2
• Never try to use SerialEM and MDS/iMDS at the same time, except as described above
• Never use SerialEM with the standard tilt holder
• It is a good idea to do a dose calibration, or better, a flatfield, before starting your session
• If beam is moving around a lot when focusing, check that pivot points are OK (or call staff)

• Set ALLOWTOPICVIEW =

-- BillRice - 28 Sep 2007