Operating F20/F30 with SerialEM

This is Chen Xu’s quick check list for the Tecnai F20 or Tecnai F30 users who already have basic knowledge of Tecnai FEG microscope: I list here the steps that I would perform if I am on the scope with cryo sample and plan to collect single particle data using SerialEM.

1. Check E-logbook to see if there have been any problems

It is not a bad idea to see if there is any problem or issue from last user that should have been logged on the E-logbook.

2. Login to Tecnai Computer

Start up Tecnai User Interface (TUI).

3. Check vacuum status

The vacuum status should show “Column Valves Closed”. IP3, IP1 and P3should all be in good ranges.

4. Check settings for H.T., Gun Lens and Extracting Voltage

Make sure they are in desired values, and set them to otherwise.

5. Cool down the scope

Add LN2 to the big cold trap dewar. This is important for cryo session.

6. Check CryoBox Position

It is a good time to make sure the CryoBox is at IN position. When it is IN, the “aperture” indicator points to position “1”, close to horizontal direction. This is absolutely needed for a serious cryo session.

7. Put in a single tilt holder with a standard grid

Currently, the standard grid is a holey grid with Au particles deposited on. Ask Chen if there isn’t one available.

8. Eucentric height, Eucentric focus and Pivot Points

This is to tune miscroscope, mainly to set beam tilt pivot points to the right level.
This should be in the order:

  1. Eucentricity
  2. precisely focus
  3. adjust pivot points using Multi-function knobs. You might want to check rotation center at this point too.

9. Check coma-free and beam conditions for high-res imaging

This is relatively more important for single particle user. Tomography user may skip this step.

  • Come-free aligning. This should be done at the highest magnification of SA range.
  • Check the shape of beam cross. This should be done at a mag higher than SA range. The bright spot should be sharp and close to the center of the whole crossed beam. Otherwise, perform gun alignment procedure. This step might also concern tomography user, as if the gun tilt is off too much the beam intensity cross gradient could cause sharp border between montage pieces.
  • Check FFT of the image from a patch of gold particles on grid.

10. Start up CCD or 3rd party software interfaces

  • On F20: DigitalMicrogaphTIASerialEM
  • On F30: Filter ControlDigitalMicrogaphSerialEM.

11. Set Bias for 4-port CCD

This is done with NO beam, and from CCD’s native controlling software interface.

  • For FEI Eagle CCD camera that was on Tecnai F20, it is from → CCD Camera → All Bias.
  • For Gatan US4000 CCD that was on F30 and now moved to F20, DigitalMicrogaphCameraSet Bias.

12. Prepare Gain Reference Files

This is done with proper beam and NO specimen. Either specimen rod is in retracted position or there is a hole or broken area that is large than the CCD area. As we mainly use SerialEM for this preparation task, this is done from SerialEMCameraPrepare Gain Reference.

For the new generation direct detector camera such as Gatan K2 Summit camera and FEI Falcon camera, the procedure to prepare gain reference is more complicated. Therefore, it is perhaps not a bad idea to use the native controlling software to prepare the gain reference. You can ask SerialEM to use that. This is done by SerialEMCamera → uncheck Normalize here.

13. Setup Low-Dose in SerialEM

A typical setup like this might work for you too.

  • View: mag = 1700x, binning = 4, area = full.
  • Focus: mag = 100kX – 200kX, binning = 4, area = half or quarter-wide.
  • Trial: mag = The same as Focus(check box “keep Focus and Trial the same”), binning = 4, area = full. The beam size should slightly smaller than CCD area so the edge of the beam can be seen on T image. This is needed for “CenterBeamFromImage” routine to work nicely.
  • Record: mag = 59,000x, binning = 1 or 2, area = full

Set proper offsets for View mag, first defocus and then IS. The IS offset only works for the specific mags of R and V. If down the road, you change the mag of V or R, you need to recheck this.

14. Cryo Holder In

Now the scope and CCD are ready, it is time to handle the real cryo sample now. I would wait until IGP1 < 10 before opening the cryo shutter.

15. Stop LN2 bubbling in the holder dewar

Allow 30+ minutes for holder temperature to settle. And then quiet the bubbling.

16. Eucentricity, without wobbling the stage if possible.

If possible, try not to use procedure that involves stage wobble. The main reason is that wobbling the stage will likely introduce LN2 bubbling again in the dewar of 626 side-entry cryo holder. The quickest way is perhaps to run Z_byVmacro in Low-Dose mode.

Example 1. Z_byV

MacroName Z_byV
##############################
# Z_byV.txt
# by Chen Xu, Feb 8, 2013
##############################
#
# a macro to adjust the eccentric center using beam tilted pairs.
# It uses Autofocus to measure the focus and adjust Z instead.
#
# assume the Eucentric Focus has been calibrated for the mag you use.

#==================================
# remember the starting focus for V
#==================================
GoToLowDoseArea V
ReportFocus
DEF = $ReportedValue1

#====================
# set objective lens
#====================
SetEucentricFocus
NormalizeLenses 2
Delay 1

#===========
# Adjust Z
#===========
Loop 2
Autofocus -1 1
ReportAutofocus
t = -1 * $reportedValue1
MoveStage 0 0 $t
echo –> Z moved $t micron
EndLoop

#==================================
# set back original defocus of View
#==================================
SetStandardFocus $DEF

17. Obtain Full Grid Overview

Go out of Low-Dose mode, go to low mag like 120x or 140x, Obj. Aperture out, set C2% to 100. Set binning_R = 4, Exposure = 0.3 second. Take a R shot to check if the image has complete full area, not partially blocked by CryoBox. It is a good time to check if the cryoBox is centered.

  • Navigator -> Open
  • Navigator -> Montaging and Grids -> Setup Full Montage
  • Response to the dialog window and define filename.
  • Click on “start” on Montage control panel.
  • Make the final overview to Map. Close the stack file.
  • Change mag back to above LM range so that Obj Lens can warm up.

Note:
As you can see, it would be nice if we can do the last three steps automatically so that we don’t have to wait for the montage to finish to increase mag. Indeed, we can by taking advantage of macro. After defining the filename for the montage, the stage will go to (0,0) by itself. You can then add current stage position as a point item and flag it with “A”, followed by running a very simple macro from Navigator -> Acquire at Points.

Example 2. TimeForCoffee.txt

MacroName TimeForCoffee
# warm up obj lens after map is done.

Vmag_LD = 2300

M
NewMap
SetMag $Vmag_LD

18. Resume Low-Dose Mode

Recheck low-dose condition, after Obj lens temperature settles down from previous step. With current geometry of the grid, set “Focus” position. Double check if the V and R mags are aligned still.
Don’t forget to put back Obj Aperture in and center it. Check the Obj stigmation at this point.

19. Mark Interested Points

Go to one of the meshes picked from grid atlas map. And you might want to “align” the images between V (e.g. 3000X) and mag at which the grid overview map is created (e.g. 140X) using “shift to marker” under navigator.

  • Adjust to eucentric hight using macro “Z_byG”. Same as before, the F should be copied as V beam temporarily.
  • Take a V shot and make it into map.
  • Add interested points as point item. And flag all of them as “A” – Acquire.

20. Check your main Macro

Make sure the main Macro and all other are loaded in Macro editors. Here is an example:

Example 3. LD.txt

MacroName LD
#main macro for data collecting in LD mode

## positioning – goto a spot
RealignToNavItem 1
Copy A P

## Center beam
T
CenterBeamFromImage
T

## Adjust Z to Eucentricity
Call Z_byG

## refine X Y
Call AlignToP

## now autofocus
G
G

## Drift Control
Call Drift

## Take shot
R
S
#=== end ====

Run this macro for one point (highlighted in navigator window). If it works fine, then the hard part is done. Congratulations!

21. Acquire at all points

Navigator -> Acquire at Points, and define the macro, in this case “LD”, as the main task.
That’s it. Sit back to watch.

22. Finishing Up

  • Close Column Valves!
  • Close the cryo shutter on the holder
  • Take out cryo holder from scope
  • Take out clamp ring and the grid from holder
  • Warm up the holder
  • Cryo Cycle
  • Log the session