Quick guide to use paceTOMO for cryo-ET data collection from Titan Krios

Load samples into the Titan. Slot 1 is reserved for cross-grating grid, which is used for daily alignment.

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.

Prepare

Software

Value	Label
serialEM	NAME
Win	OS_NAME
17763.3287	OS_VERSION
Regents of the University of Colorado	DEVELOPER
https://bio3d.colorado.edu/SerialEM/download.html	LINK
4.1.0_beta_9.64	VERSION

Start server from icon on the desktop

Make a new folder in X drive for data collection

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

To launch

Software

Value	Label
serialEM	NAME
Win	OS_NAME
17763.3287	OS_VERSION
Regents of the University of Colorado	DEVELOPER
https://bio3d.colorado.edu/SerialEM/download.html	LINK
4.1.0_beta_9.64	VERSION

Open the Anaconda shell on the K3 PC desktop.

Use terminal with administrator privilege, then run the following command to start the software:

#start serialEM (Anaconda shell)
conda activate serialem
cd C:/Users/VALUEDGATANCUSTOMER/Documents/SerialEM_beta
SerialEM.exe

in serialEM, open the newly copied settings file as well as the Navigator. Load 580x from imaging states window

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”).

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.

Move stage to hole position.

Initial configuration of Record settings:

Change IS to desired low dose data collection settings (either 42k or 53k). Double click to activate the preset.

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.

Check Record beam centering on flu-screen and check flux in GMS3 with a one-second exposure.

Update hardware dark reference:

In GMS3, find Camera tab > Prepare Dark Reference.

Takes ~10 seconds.

GIF tune:

Check beam centering on flu-screen and change spot size to achieve 50 e/p/s flux. Lift screen and unblank beam.

Perform full GIF tune in GMS3 with illumination settings intended for SerialEM data collection.

Prepare gain reference if necessary. (typically needed once a week)

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.

If gain reference is needed, select gain reference from GMS3 Camera drop-down tab and proceed with the prompts.

In TEM UI, move stage to foil position.

Check View magnification and centering on Flu screen.

If changing anything, tick and untick continuous update in serialEM

Find eucentricity with SerialEM Rough Eucentricity function. Can be found in the drop-down menu.

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.

Use SerialEM to autofocus to -2.5 um.

Correct astigmatism by CTF in SerialEM.

Coma-free alignment by CTF in SerialEM.

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).

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.

Align SerialEM Record/Focus/View offsets:

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.

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.

With the cross-grating loaded, switch to 135x Atlas IS.

Set up full montage (6x9 atlas) in serialEM drop-down menu. Run MapGrids macro from script editor.

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

Optional step: if lamella is being imaged and paceTOMO is not used, pre-tilt determination is required ahead of time.

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.

Obtain square/lamella detailed montage/maps using los-does presets (42k or 53k)

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.

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).

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.

Inspect detailed square/lamella montages for data collection.

Under Camera setup in serialEM, check exposure and frame time depending on desired total dose.

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.

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.

Add points for details of interest in the navigator. Save it when done.

Under Low Dose control in serialEM, ensure Focus position on tilt axis is 0, i.e. the same area as Recording region.

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.

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.

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

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.

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.

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.

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.

Put in the number for tilt angles and steps based on the estimated pre-tilt.

Once done, Save the targets file and close it.

Repeat above steps for each PACE group.

Pre-run checklist:

Focus area 0 microns away.

Untick "stage shift for large mouse shifts".

OA inserted and Slit is in

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.