Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Register use with pHp
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open vacuum overview (bottom right)

• • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retrieve CCDBlankEnable Box

Check for beam and alignment before loading sample (NYSBC Staff)

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu

• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retrieve CCDBlankEnable Box

Check for beam and alignment before loading sample (NYSBC Staff)

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Tecnai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked

Check for beam and alignment before loading sample (NYSBC Staff)

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Tecnai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked

Check for beam and alignment before loading sample (NYSBC Staff)

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Tecnai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked

Check for beam and alignment before loading sample (NYSBC Staff)

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu

• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked

Check for beam and alignment before loading sample (NYSBC Staff)

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup , MinghuiHu

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam and alignment before loading sample

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Acessing status of the FEG

• Open Tecnai User Interface
• Go to Setup/FEG Control panel
  • If the operate button is not yellow FEG is off
  • Call NYSBC or FEI staff

Keeping the beam centered when raising screen (as opposed to offsetting for the camera):

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam and alignment before loading sample

• Insert specimen holder to first stop
• In setup menu open column valves when option becomes available
• Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)
• Warm up cryoholder if used
• Log out of computers and turn off monitors

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

• TEM data computer controls Fast Scan CCD holds data and provides access to internet
• FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam and alignment before loading sample

• • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam and alignment before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves when option becomes available
  • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam and alignment before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves when option becomes available
  • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Sample Insertion

• Have NYSBC staff insert your sample

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam and alignment before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves when option becomes available
  • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Check Condenser Aperture Astigmatism
  • Bring beam to crossover; if beam is not round
  • Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
  • Use multifunction knob to make beam round
  • Unclick Stigmator button

Specimen Imaging

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Focus

• Press Eucentric Focus on Right control panel
• Move Z +/- to obtain focus
  • if you are having trouble seeing thon rings dropping the magnification may be helpful

• Alpha Wobbler method
  • in Search mode
  • press alpha wobbler Left control panel
  • Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Reset holder
• Remove objective aperture
• Remove sample
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Computer Crash

• Close gun valve manually
  • This is located on the left side of the viewing port labeled "Close V7"
  • If you manually close the valves you must reopen them before clicking "Open Column Valves"
• Restart computer
• Logon as Administrator
• Goto: My Computer
  • right click and select manage/services and applications/services/TEM server
  • Press start
• Logon as yourself

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register film number with pHp
• Log on to TEM control computer (right two screens)

• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

• • Insert specimen holder to first stop

• • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search

• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly

• • Stigmator menu should pop up
  • Make sure Condenser button is selected

InsertRoomTemperatureSample

InsertCryoSample

• Check vacuum IGP1 should be max 15
• Open column valves

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

• Move Z +/- to obtain focus

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves

• Remove objective aperture

• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Computer Crash

• Close gun valve manually
  • This is located on the left side of the viewing port labeled "Close V7"
  • If you manually close the valves you must reopen them before clicking "Open Column Valves"
• Restart computer
• Logon as Administrator
• Goto: My Computer
  • right click and select manage/services and applications/services/TEM server
  • Press start
• Logon as yourself

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)

• • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search

• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Computer Crash

• Close gun valve manually
  • This is located on the left side of the viewing port labeled "Close V7"
  • If you manually close the valves you must reopen them before clicking "Open Column Valves"
• Restart computer
• Logon as Administrator
• Goto: My Computer
  • right click and select manage/services and applications/services/TEM server
  • Press start
• Logon as yourself

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Computer Crash

• Close gun valve manually
  • This is located on the left side of the viewing port labeled "Close V7"
  • If you manually close the valves you must reopen them before clicking "Open Column Valves"
• Restart computer
• Logon as Administrator
• Goto: My Computer

• • Press start
• Logon as yourself

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • Start Low Dose mode before aligning the beam or alignments will be off.
  • If no beam go to Focus and lower magnification, do not adjust the beam in Search

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • Start Low Dose mode before aligning the beam or alignments will be off.

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves

• • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in em-signup database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Instructions to Locate Beam

Instructions to Find CCD Blank Enable

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get close to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Common Problems

Keeping the beam centered when raising screen

• Workspace Layout
  • Detector Config
  • Uncheck “Auto Mode”

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder
• CCD Blank enable should be on screen with box checked
  • Instructions to Retreive CCDBlankEnable Box

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read ~3900-3950
• Accelerating voltage should be 200kV
• Click Prepump airlock (setup menu)
  • While airlock is prepumping load sample in holder

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Get clost to focus with focus knob (right panel)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)

• Accelerating voltage should be 200kV

• • While airlock is prepumping load sample in holder

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
  • If necessary, CreateNewUser^?
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read 3900
• Accelerating voltage should be 200kV
• Activate turbo pump on airlock (setup menu)
  • While airlock is prepumping load sample in holder

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Turn off airlock turbo pump
• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Click Eucentric focus (right control pad)
• Find focus with focus knob (right control pad)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)

• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read 3900
• Accelerating voltage should be 200kV
• Activate turbo pump on airlock (setup menu)
  • While airlock is prepumping load sample in holder

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Turn off airlock turbo pump
• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Click Eucentric focus (right control pad)
• Find focus with focus knob (right control pad)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005

Tecnai F20 (basic operations)

TEM data computer controls Fast Scan CCD holds data and provides access to internet FEI control computer controls EM and Slow Scan 4k CCD camera and runs tomography software

Press Scroll Lock 2x to switch screens

Contents

Initial Setup

• Log on to TEM data computer (left screen)
• Register use with Grey matter
• Log on to TEM control computer (right double screen)
• Start TCL
  • Click start in dialog box
  • EMMENU (pull down)
  • Select Start EMMenu
• Open Technai User Interface
• Open vacuum overview (bottom right)
  • IGPI should not read over 15 (6 best value)
  • P3 indicates moisture of film (60 new film-20 dry film)
• Extraction voltage should read 3900
• Accelerating voltage should be 200kV
• Activate turbo pump on airlock (setup menu)
  • While airlock is prepumping load sample in holder

Check for beam before loading sample

• • Insert specimen holder to first stop
  • In setup menu open column valves
  • If no beam lower magnification

InsertRoomTemperatureSample

InsertCryoSample

Setup Beam

• Turn off airlock turbo pump
• Check vacuum IGPI should be max 15
• Open column valves
• Obtain rough focus
• Condenser Aperture Alignment and Stigmation
• Check condenser aperture size (plastic sections-#4)
• Check alignment of condenser aperture
  • At cross over center beam with beam shift (left control pad)
  • Spread beam and center with aperture X, Y
  • Repeat until beam spreads evenly
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Condenser button is selected
• Bring beam to crossover
• Use multifunction knob to make beam round
• Unclick Stigmator button

Objective Aperture Alignment and Stigmation

• Check Objective aperture size (plastic sections-#3)
• Put in objective aperture
  • Press diffraction (right control pad)
  • Condense diffraction spot
• Center objective aperture around the spot with the aperture controls X, Y
• Press Stigmator button on left control panel
  • Stigmator menu should pop up
  • Make sure Objective button is selected
• Using CCD camera and Power spectrum bring image to focus
  • If Thon rings are out of round fix with multifunction knobs
• Unclick Stigmator button

Adjust Eucentric Specimen Height

• Click Eucentric focus (right control pad)
• Find focus with focus knob (right control pad)
• Go to low magnification
• Click Alpha Wobbler L2
• Move Z +/- to obtain focus

A few words about focus

• If you are using the power spectra (easier to use at higher magnifications)
• Over-focus has a bright center with division ring
• Focus has a bright center that lessens in intensity as it moves from center
• Under-focus has a bright center with division rings forming from the outside
• The amount of under focus used for an image is directly dependant on the size of the feature of interest.

Low Dose

• Click low dose tab
• Use search to find area of interest
• Use focus to focus area of interest
• When ready to image use exposure mode

TomoGraphy

Taking a FilmImage

Taking a CcdImage

Shut down

• Close column valves
• Turn on turbo
• Remove objective aperture
• Record date and ending plate number in Grey Matter database on TEM data computer

End of day shutdown

• Remove Dewar for cold trap and place the cup under the coils
• Run cryo cycle (setup menu)

Visit the Cryo EM Web site at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 18 Mar 2005