A simple guide to basic Titan Krios operation zhy, jpg& vdb2110, vdb2014

To start a new session

1. Log in using personal username and password

2. Start TEM User Interface

3. Start FluCam Viewer, insert screen

4. Start Filter Control (wait until fully loaded)

5. Start Digital Micrograph (wait until fully loaded)

6. Check Vacuum (log): Gun =1, Octagon =1 and Liner <15 at liquid nitrogen temp.

Gun =1, Octagon <=18 and Linear <=18 at room temperature

7. Open Column valves.

8. Insert Screen & Check for beam on the screen. If not visible:

lower magnification,
lower spot size number,
remove objective or selected area apertures,
condense beam with “Intensity” knob,
move stage (check for grid bars or thick ice)
if still no beam, close gun valves,remove sample, and check again
When beam is visible, move on to step 9

9. Prepare to dock new cassette & load new cartridges

close column valve
turn on autoloader turbo pump= always on (if not on already)

Docking a new cassette in the autoloader

1, Make sure column valves are closed

2. Set Turbo pump to always on (autoloader flap out page)

3. Prepare to dock cassette in autoloader

a) check indicator pin on top of precooled nanocab -make sure it is not frozen & can pop up

b) cover top of nanocab with aluminum foil to keep it frost free during transfer from workstation

4. Slide nanocab onto bottom of autoloader

round side goes in first, toward column
should hear a soft click as it activates the switch
click DOCK button on user interface

5. Wait! -Several minutes

1. liquid nitrogen may overflow (normal)

2.Caution- it squirts out the front!

6. Vacuum and Temperatures must be checked before proceeding

autoloader vacuum must be below 25log (better to be 22 or lower)
cartridge gripper temp <-175C
cassette gripper temp <-154C

6. Perform Inventory- inventory whole cassette prior to starting

1. Click Inventory button

2. Wait

3. Don't interrupt inventory in progress, make hang program

7. Load cartridge

cartridge may be loaded from any occupied slot
blue background means that position has a cartridge
yellow background means that cartridge is on the stage
outlined background means that position is selected for action
greyed out background means cartridge can’t be loaded right now
green number means that cartridge has been inventoried by the autoloader

Survey a grid and store positions for regions of interest

1. Lower magnification

use 100x or 81x mag
allows for viewing large overview of the grid
need objective aperture OUT

2. Insert screen

3. Prepare to open column valve

check vacuum of gun = 1 log
liner = 15 log (or 18- 22 at room temp)
octagon = 1 log
when ok, open column valve

4. Controlling Stage travel

1. Open the Stage2 control panel (lower right pop up menu)

2.double click any spot on stage display to move there automatically

3.or move the stage joystick manually

4.survey the quality of grid squares

5. store interesting square positions (good ice etc.)

a) naming them is useful

b) avoid confusing numbers that conflict with position numbers

6. store position of an empty area (for dose calibration and GIF tuning).

5. To start data collection from a square

1. increase magnification to ~2000x

2. use High Contrast button on the FluCam Viewer if sample is low contrast

6. adjust eucentric height

1. regions of interest should always be adjusted to eucentric height before data collection no matter what type of data is collected (tilt series or single particle images)

2. the calibrations & alignments of the microscope are done at eucentric height

3. magnification, focus and parallel beam ranges are only correct at eucentric height

4. update this position or add as a new position in Stage2 (now correct z height is stored too)

5.check objective astigmatism after moving from one area to another

6. Control panels

1. All available control panels are accessible either by quick link (the pop up menu on the bottom right on the User Interface)

2. or browsing through the workset tabs

3. if a control panel does not appear in the pop up list, it may already be open on the desktop

Preparing CCD and Autofilter (Energy Filter for EFTEM)

1. Find an empty square

From stored positions on Stage2, select the one for an empty square and click “Go to”
If none are empty, a large hole may be used

2. center beam

1. insert flu screen

2. go to desired data collection magnification

3. Center beam and expand beam to cover screen.

3. choose data collection camera

1. go to CCD/TV Camera control panel

2. select camera (CCD or GIF CCD).

4. Adjust imaging settings for different modes

1. go to CCD/TV Camera control panel, expand flap out and check the settings for “Search”, “Preview” and “Acquire”

2. Change the settings (mode, bin and exposure time) as needed.

3. A good example:

Search button = continuous readout, high binning (8), short exposure (0.062 s)

Preview button = continuous readout, less binning (2), longer exposure (0.5 s)

Acquire button =single image, bin=1, 1 second exposure

5. Retract screen

6. Check beam alignment with CCD

1. click “Search” button on CCD/TV Camera control panel

2.an image of the beam should show up in Digital Micrograph

3. re-Center and re-expand the beam on CCD if edges of beam is seen.

a)The red circle on the flu screenmarks the center of the 4k x 4k

b) The green circle marks the center of the GIF (note that it is slightly off center)

4. Expand the beam more until counts are lowered to the desired data collection range

5. Check Beam Settings to make sure beam still in parallel range

7. Check Gain Reference

1.Take a CCD image of the beam (no sample) to check for uniformity

2. If not uniformly grey, a new gain reference is needed

3. Acquire a new gain reference

1. use a target count roughly equal to the one you are using to collect data (2000-4000)

2. average 4-10 frames)

4. take a new (gain subtracted) image of the beam- it should now be uniform

5. It may not be necessary to generate a new gain reference for every session.

a) It is good to always collect one image with a weak beam to check the quality of the image

b) Then decide whether or not to generate a new gain reference.

For EFTEM with GIF CCD

8. insert Flu screen, center beam around the small green GIF circle.

9. Select GIF CCD

go to Autofilter control panel
Click on EFTEM
the camera should automatically change to GIF CCD on the CCD/TV Camera panel
If not, choose it manually

10. Repeat steps4-7 above for the GIF CCD.

11. Choose the energy slit size to use

a) on Filter Control work page, type in 30 eV slit width (50eV may also be used)

b) then check “slit in” to insert a 30 eV energy slit.

12. press “Align ZLP” on the Autofilter control panel to align zero loss peak.

13. insert Flu screen

14. center beam to GIF camera

a) raise magnification to the highest SA magnification

b) Center beam around GIF circle.

15. Tune the GIF (Gatan energy filter )

a) click “Tune GIF” in the Antofilter control panel

b) watch the progress in Digital Micrograph

c) adjust beam intensity when necessary by expanding or condensing the beam with the “Intensity” knob.

16. lower magnification to data collection mag.

17. There should be no need to tune GIF more than once per session

18. Align the ZLP often, it may drift depending on the stability of the temperature and the environment,

Final check list just before collecting data

1. Eucentric height is checked/adjusted for each stored specimen location.

2. Turbo pump for autoloader is off

3. objective aperture is inserted and centered

4. objective lens astigmasm is checked and corrected

5. direct alignment (beam tilt pivot point, rotation center or coma-free alignment) is done

6. low dose is on (for low dose tilt series with FEI tomo)

7. 30 eV energy slit is inserted for EF tomo with GIF.