TITLE: Quick guide to use paceTOMO for cryo-ET data collection from Titan Krios
AUTHORS: Josh Hutchings, Siyu Chen

[DESCRIPTION]
This quick guide provides key minimal steps for preparing the Titan/SerialEM for tomogram data collection on lamella or in vitro specimens with K3 camera. paceTOMO routine is also included for a typical tomogram data collection session.
Please note that this is not an exhaustive guide, but summarises order of key steps.

[BEFORE_START]
Check the following before the session is started:
Titan LN2 tank at least 20% full.
GMS3 should be open on the K3 PC. If not, open it (takes few minutes).
Check that GMS3 is in Power-User mode (Help menu).
Cross-grating grid should be on the stage.
 



[GUIDELINES]
Useful resources: 
SerialEM repository (Nexperion): https://serialemscripts.nexperion.net/
SerialEM mailing list: https://bio3d.colorado.edu/SerialEM/#List
All SerialEM help dialogues can be accessed from SerialEM directly

[STEPS]
SECTION: paceTOMO data collection workflow
7. Initial configuration of Record settings:
SECTION: paceTOMO data collection workflow
6. Move stage to hole position.
SECTION: Preparation before performing alignment
2. Do inventory in TEM UI once all temperatures shown on the screen are <100K. If not already, load the cross-grating grid onto the microscope.
SECTION: Preparation before performing alignment
1. Load samples into the Titan. Slot 1 is reserved for cross-grating grid, which is used for daily alignment.

 

SECTION: Preparation before performing alignment
5. Decide on CDS or non-CDS mode for the data collection session in GMS3 and check that the SerialEM camera set-up is consistent with that. 
CDS mode mitigates some K3 gain artefacts but will increase the data acquisition time.
SECTION: Preparation before performing alignment
3.1. Start server from icon on the desktop
SECTION: Preparation before performing alignment
3. Prepare 
 

SECTION: Preparation before performing alignment
3.2. Make a new folder in X drive for data collection
SECTION: Preparation before performing alignment
3.3. Find a setting file that has been recently used and start from this file: 
The master settings can be found either from other people or your own previous settings. Copy such setting file and paste it to the current data collection folder, then rename it to a unique, informative name. For example: 20230101_name_from_name_20221231_settings.txt

SECTION: Preparation before performing alignment
3.4. To launch 
 

Open the Anaconda shell on the K3 PC desktop. 
Use terminal with administrator privilege, then run the following command to start the software:
 

SECTION: Preparation before performing alignment
4. With flu-screen down (L1 from the control pad, or in GMS), find a completely empty square (save position as e.g. “hole”) and a square with intact film (save position as e.g. “film”).
 

SECTION: Preparation before performing alignment
3.5. in serialEM, open the newly copied settings file as well as the Navigator. Load 580x from imaging states window
SECTION: paceTOMO data collection workflow
7.1. Change IS to desired low dose data collection settings (either 42k or 53k). Double click to activate the preset.
SECTION: paceTOMO data collection workflow
7.2. Note the C2 aperture (typically 70 or 100 mm can be used) - this will influence the flux.
Check View setting magnification (generally 4800x or higher) and slit is inserted for Record mag.

SECTION: paceTOMO data collection workflow
7.3. Check Record beam centering on flu-screen and check flux in GMS3 with a one-second exposure.
 

 

 

SECTION: paceTOMO data collection workflow
8. Update hardware dark reference:
In GMS3, find Camera tab > Prepare Dark Reference.
Takes ~10 seconds.
SECTION: paceTOMO data collection workflow
9. GIF tune:
SECTION: paceTOMO data collection workflow
9.1. Check beam centering on flu-screen and change spot size to achieve 50 e/p/s flux. Lift screen and unblank beam.
 

SECTION: paceTOMO data collection workflow
9.2. Perform full GIF tune in GMS3 with illumination settings intended for SerialEM data collection.
 

SECTION: paceTOMO data collection workflow
10. Prepare gain reference if necessary. (typically needed once a week)

SECTION: paceTOMO data collection workflow
10.1. It is recommended to check if the reference is needed. To perform that in GMS3: 
Take a dark reference
adjust the beam intensity go to to ~15eps
take an 1s, 20-frame exposure
in the Process tab, click autocorrelation and see if there is any pattern existing.
SECTION: paceTOMO data collection workflow
10.2. If gain reference is needed, select gain reference from GMS3 Camera drop-down tab and proceed with the prompts.
 

 

SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
11. In TEM UI, move stage to foil position.
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
12. Check View magnification and centering on Flu screen. 
If changing anything, tick and untick continuous update in serialEM
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
14. Switch to Record magnification and find focus with GMS3 (with Live > FFT window on). When FFT doesn't show any Thon rings, press L2 on the control panel to zero defocus.
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
13. Find eucentricity with SerialEM Rough Eucentricity function. Can be found in the drop-down menu.
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
15. Use SerialEM to autofocus to -2.5 um.
 

SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
16. Correct astigmatism by CTF in SerialEM. 
 

SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
17. Coma-free alignment by CTF in SerialEM. 
 

SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
18. Coma vs Image Shift calibration.
 
When the task is done, put in the C2 aperture size: 70 or 100 (check Apertures tab in TEM UI).
SECTION: Aquire multiple atlases and screen the sample quality
24. Optional step: if lamella is being imaged and paceTOMO is not used, pre-tilt determination is required ahead of time.
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
19. Insert OA (objective aperture, 70 or 100 um) and check centering in diffraction mode (make sure the flu screen is DOWN). 
Use roll wheel to get bright view and use rings to center OA. 
Change magnification to meters when rings are too small to be visualized.
 
Once done, correct astigmatism by CTF in SerialEM again.
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
20. Align SerialEM Record/Focus/View offsets:
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
20.1. Check “continuous update” in SerialEM for the low-dose IS.
in serialEM, copy Record setting parameters to Focus and Trial settings.
Lower down the flu-screen to check centering of Record/Focus/View settings.
SECTION: Align the microscope with stable carbon/gold materials in the field of view (over the foil)
20.2. Check image shift for Record/View settings using a feature such as a bead on the XG grid. 
Untick “move stage for big mouse shifts” and retick once offset is set.

 
Once done, uncheck “continuous update”, save settings and update IS.
SECTION: Aquire multiple atlases and screen the sample quality
21. With the cross-grating loaded, switch to 135x Atlas IS.
SECTION: Aquire multiple atlases and screen the sample quality
22. Set up full montage (6x9 atlas) in serialEM drop-down menu. Run MapGrids macro from script editor.
 

SECTION: Aquire multiple atlases and screen the sample quality
23. Inspect each atlas and decide on overall grid quality. Reloading grids will require registration alignment to account for small shifts/rotations during reloading. 
 

Change back to 135x first for all of the following alignments
SECTION: Aquire multiple atlases and screen the sample quality
24.1. Tilt stage to +40’ and take a Record image. 
Use shift+drag-left-click to measure top-to-bottom lamella length (appears in log window). 
Tilt stage to -40’ and take a Record image. 
Use shift+drag-left-click to measure top-to-bottom lamella length (appears in log window).
Whichever gives the longest distance is the direction of the pre-tilt (e.g. +/– 9 to 18’). 
Tilt stage to desired pre-tilt and stay at this angle for subsequent steps. 
*for PACEtomo routine, change the tilt back to 0° since the script calculates the tilt by itself.
SECTION: Aquire multiple atlases and screen the sample quality
27. Use sacrificial area on the specimen to check defocus (with target something like 4-5 um) and View/Record image shift before queueing up for data collection.
Use vacuum area next to data collection area to re-check beam centering and ZLP.
SECTION: Aquire multiple atlases and screen the sample quality
25. Obtain square/lamella detailed montage/maps using los-does presets (42k or 53k)
SECTION: Aquire multiple atlases and screen the sample quality
25.1. Insert objective aperture and check slit is inserted.
Switch to LD data collection IS (the same used for XG calibrations above) and do shift-to-marker with View using one or two square/lamella maps. 
 

SECTION: Aquire multiple atlases and screen the sample quality
25.2. Load each square/lamella map in the Navigator and add a point in the centre of the square/lamella for montage view if desired.
Set each points to “Acquire” and select “new file at item” with montage settings (provide an indexable name, e.g. squarev1.mrc). 

 

	
SECTION: Aquire multiple atlases and screen the sample quality
25.3. in serialEM drop down menu, select Navigator > Acquire at items. No tasks checked except for montage/map collection. 
Make sure Z height is applied by unchecking the mark. 
Use LD View for montage and use stage movements. 
Save Navigator when done.
SECTION: Aquire multiple atlases and screen the sample quality
26. Inspect detailed square/lamella montages for data collection.
SECTION: Aquire multiple atlases and screen the sample quality
26.1. Under Camera setup in serialEM, check exposure and frame time depending on desired total dose.

 


SECTION: Aquire multiple atlases and screen the sample quality
26.2. In the same tab, set folder for frames to be saved to.
Also set file saving options: tif (LZW compression), set filename as current open file (minus extension), sequential number starting from 0, tilt angle in name.
SECTION: Aquire multiple atlases and screen the sample quality
28. Add points for details of interest in the navigator. Save it when done.
SECTION: paceTOMO data collection workflow
29. Under Low Dose control in serialEM, ensure Focus position on tilt axis is 0, i.e. the same area as Recording region.
SECTION: paceTOMO data collection workflow
30. Decide on camera and frames settings:
Focus, Trial and Preview exposures can be set for ~ 1 e/A2, e.g. ~0.2s if dose is around 5e/A2/s
View and Preview should have the same binning, normally 4x.
Decide on Record exposure and number of frames, ideally at least ~ 0.4 e/A2/frame.
Ensure the View defocus offset is 50 microns, rather than 150+ microns.
Record file saving options should have same root as open file (minus extension), with tilt angle and navigator item number included.
SECTION: paceTOMO data collection workflow
31. Under Camera Setup, change the frames folder to a dedicated folder for PACE and ensure dose fractionation settings are as desired.

 

Make another directory dedicated to PACEtomo targets
Check Tilt Axis Offset under the "Tasks > Eucentricity" tab in serialEM is set to the calibrated value, normally -2.2 microns. This is important for a stable defocus across the tilt series.
SECTION: paceTOMO data collection workflow
32. Pre-run checklist:
Focus area 0 microns away.
Untick "stage shift for large mouse shifts".
OA inserted and Slit is in
SECTION: paceTOMO data collection workflow
31.1. Check desired settings in the PACEtomo.py and PACEtomo_selectTargets.py scripts.
No need to change most of the things. Check to make sure:
zeroExpTime is set to 0, unless hybrid scheme is desired.  

In PACEtomo.py:
startTilt and max tilt can be changed later.
minDefocus, maxDefocus can be -3 to -5 but can also modify later 
delayIS and delayTilt should be 0.5 or even 1
taOffsetPos is something like 0.8, based on Josh’s benchmarking on our Titan1 to deal with systematic defocus increase at high tilt

In PACEtomo_selectTargets.py:
Guidance is true for guided anchormap routine
beamDiameter should be something like 1.8 for 42x and suggested dose
maxTilt is ~60° for a 37-tilt tomogram
SECTION: paceTOMO data collection workflow
31.2. Inspect each lamella/square montage and decide on the number of needed PACE groups.
Think diligently about how to arrange TS and where to place the sacrificial focus/tracking area - preferably something distinct. One can use Navigator Points on montages.
 


SECTION: paceTOMO data collection workflow
31.3. For each PACE group, add Navigator points for intended acquisition areas. As always, think diligently above the beam diameter and spacing of acquisition areas.
 
The first point in the group must be the focus/tracking area and have enough features, e.g. a mitochondrion, a multi-lamella body, bacterial cell, etc.
SECTION: paceTOMO data collection workflow
31.4. Select this first point in group and move stage to XYZ in navigator. Autofocus to -4 μm here.
Make sure "move stage for big mouse shifts" is unticked.
Verify grouping by clicking 'collapse' in the Navigator and checking the desired number of acquisition points, including the focus/tracking position.
With the first item in the group highlighted, run PACEtomo_selectTargets from the editor and follow the prompts.
 

SECTION: paceTOMO data collection workflow
31.5. In the targets GUI, assign at least three additional focus positions with mouse middle-clicks in vacant areas, hit "measure geometry" and wait. Note the estimated pre-tilt and rotation for this group.
SECTION: paceTOMO data collection workflow
31.6. Put in the number for tilt angles and steps based on the estimated pre-tilt. 
 
Once done, Save the targets file and close it.

 

SECTION: paceTOMO data collection workflow
31.7. Repeat above steps for each PACE group.
SECTION: paceTOMO data collection workflow
32.1. Go to "Navigator > Acquire at Items" to run the PACEtomo script at each group. Turn off defocus cycling/focus and make sure "Manage Dewars" is always checked.