Return to Cryo EM web page at http://www.nysbc.org/facilities/CEM

To make a montage of your grid

1. Set magnification to 170X, maximum beam spread.
2. Change spot size 5 (with the console buttons, check Tecnai interface on left screen)
3. Go to Camera&Macro Controls Setup/Record tab Bining: 8 Exposure: 0.1 second
4. Make sure to increase registration number if you intend to transfer points from previous montages
5. Go to Navigator Menu/Open, then Navigator/Montage/Super Montage/Setup Full Montage, Click OK
6. For the header of the montage file select all the available options
7. 10 % overlap approximately 11x11 pieces. Larger files will crash in loading.
8. Go to Montage controls and click Start (to start the montage)
9. Save the montage as a (.st) stack file, double check the file extension (.st).
10. Save navigator file as .nav
11. When done, it will ask if you want to make a new map. ALWAYS SAY YES
12. Choose file-->close, otherwise you will not be able to change camera settings. This only closes the file for writing.

Low Dose Montage

1. Go to exposure mode in Tecnai Low Dose
2. Open SerialEM
   • Once SerialEM is open do not use Tecnai interface to control microscope
3. See above instructions
4. After collecting montage pick points, move to area and save point in the Tecnai Specimen Position feature
5. Close SerialEM
6. Resume normal Low Dose operations

Aligning rotated montage in Navigator

1. Increase registration
2. Collect new montage with identical parameters
3. Select a minimum of 3 points on both maps
   • Magnifying the image when picking points is helpful to make the marked points more accurate.
4. Go to each point and check Registration Point box on top of Navigator panel.
   • 1R1 = 2R1, 1R2 = 2R2, etc
5. Go to Navigator on pull down menu and select Transform Items

Predosing Sample

• Go to 150 micron Condenser 2 aperture #4
• Obtain a new Gain Reference to calibrate dose
  • Take record image use ctl + i to get stats
  • Camera/Prepare Gain Ref...
  • Enter mean value in target dose box
• Add stage positions of areas to does
• Check Acquire box for each point
• Navigator/Acquire at points...
• Select Macro 4 to dose 2000e-
• Select Close Column Valves when done

Tomogram settings

• File/close (to close montage image stack)
• Go to Setup menu/Record Bining: 2 Exposure: 1 second

To Gain with Camera

• Go to Camera/Gain Ref Policy(default4900)

To start a Tilt Series

1. Go to Tilt Series menu/Setup/Start
2. Select desired parameters

To finish a Tilt Series

1. After the tilt series is done go to Tilt Series menu/Terminate
2. Select "Yes" to close image stack
3. Select "No" to save 90 degrees rotated image in Buffer H

To start next Tilt Series

1. Go back to 0 tilt (on tilt control menu)
2. Go to next area (predose if necessary)
3. If you've changed magnification, go back to desired mag
4. Do autofocus
5. Start new tilt series

Useful Tip

• If you want to go to a particular slice of your stack file go to the File menu/Read/select file/select slice

F20 Defocus table for common magnifications

• When changing the binning value in Camera&Macro Ctrls after taking a full montage at 170x, don't forget to change the exposure time to 1 sec
• Set ALLOWTOPICVIEW =