Difference: TietzTomography (5 vs. 6)

Tomography using TVIPS EM Menu 3.0

Contents

Check Eucentric Focus

• 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

Microscope alignment

• Essential to functioning of Autofocus and tomography in general found in Alignments workspace under Tune tab
• Gun alignments (tilt and shift)
• Beam alignments (rotation center, pivot points, HM Beam)
• Img alignments (HM Beam)
• Skip cosmetic alignments and calibrations

Update Flat Field images (if necessary)

• Remove sample to first stop or Trash.findDFdf a hole
• Use "Continuous Readout" to set illumination
  • End with spacebar
• Provide stong beam for ~4000 counts average
• Click Create Flat fields
• Check data to make sure it is reasonable

Calibrate focus (always necessary)

• Set up high contrast image with fairly bright beam (gold particles good) close to focus
• Click Auto focus
• Adjust camera parameters for strong exposure 2x2 binning (e.g. 4000 counts)
• click Calibrate
• check calibration by selecting particular focus on upper left
  • check power spectrum and fitctf rings if >29k
• Run for all magnifications you will be using
  • exposure and focus positions should be at the same mag so no focus differential
  • also no mag changes between focus and exposure (e.g., if tracking is done in between)
• If blurred image is obtained during autofocus
  • could be image movement due to EM slow response
  • Go closer to focus
  • Weaken beam and lengthen exposure
  • Check focus

Start Tomography program

• Start TCL
• Select EMMENU in pulldown
• Click Positions
• Read extensive Help section in EM Menu about tomography

Update Calibrations (if necessary)

• Click Calibrations
• Check dates of calibrations at all mags to be used
  • Click Update TEM after changing mag to get dates
• ensure high contrast area with fairly bright beam and stable specimen
  • Top to bottom, click buttons
  • Calibrate for all mags to be used

Check Tilt Range

• Locate area of interest
• Open Control TEM on EM Menu
• Check either tilt extreme
• Can also use TEM control feature in Technai User Interface

Set up Tomography

• Refer to extensive Help section in EM Menu about tomography
• In Positions dialog box
• Calibrations are already stored and do not need to be repeated camera has been moved or goiniometer has been worked on
• Click Show Positions button to check parameters for each of the 4 "Positions"
  • Set camera parameters for each position (format and exposure)
  • Set defocus under Focus tab
• Click apply after any change
• Go to lower magnification (~ 4 clicks)
• Update Scanning parameters and copy to Search
• Go to Exposure Tracking Focus and adjust parameters
  • Press Update Positions when done
• Check each position with exposure at maximum angles before beginning series
• Check Lock Position box
• Before collecting series, click center detail

Low Dose Tomography Setup

• Go into Scanning position
• Click Areas button
  • Allows definition of location of Tracking and Focus positions along the tilt axis
• calculates offset of image shift
• requires manual adjustment of beam shift
  • uncheck Lock Positions
  • Goto each position (Exposure, Tracking, Focus, Search) and adjust beam position
  • click Update Positions
• Do not run Areas again
  • The image shift dislocations become additive, but not shown as additive

Tilt Series

• If start angle is positive angle increments need to be negative
• If using Saxton Screen, start angle needs to be positive
• Check tracking box for all images
• Do not check Focus box for all images (about every third)
• Do not check search box for any images
• Press Start

Displaying series

• Click Open series in EM Menu
• Use Series button to display as a movie

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

-- DavidStokes - 18 Mar 2005

• Set ALLOWTOPICCHANGE = CemfacGroup