LowDose Subtomo
Goal:
- Setup low dose modes.
- Set camera parameters for all modes.
ALWAYS PUT THE SCREEN DOWN WHEN CHANGING OPTICS ON the K2, otherwise you will burn the camera!!!
We should still be in Nanoprobe mode.
SerialEM general settings
- Buffer controls, Roll Buffers A -> H, nothing else selected:
- Image Alignment & Focus control panel: check if "center image shift on tilt axis" is selected.
- Image Alignment & Focus control panel: press ResetImageShift to apply this important additional shift.
.
- Low Dose Control: switch on Low Dose Mode, select "continuous update of mag & beam".
- Low Dose Control: deselect Rotate inter-area axis so Trial and Focus are positioned along the stage tilt axis.
Record mode setup
- WITH THE SCREEN DOWN,
Low Dose Control: "Area to show when screen down" select Record and
adjust the beam and magnification with the Titan hand panels.
- We mostly use Nanoprobe spot 6 or 7, aim for a camera dose rate of ~15 e/pixel/sec with the sample in the beam, the objective aperture inserted and the energy slit inserted.
- If necessary, center beam in Titan UI with Direct Alignment/Tomo Beam shift.
- Go to an empty area.
- Take a Preview exposure.
- SerialEM menu Calibration, Electron Dose:
Focus/Trial/Preview mode setup
Focus, Trial, Record and Preview will get the same optical settings:
- Low Dose Control: while in Record mode, click on T and F in "Copy current area mag & beam to".
- Make sure a 20 eV slit is inserted for all modes!
View mode setup
- For View mode set magnification 15K, same spot size as used for the other modes , -100 micron defocus.
NOTE: any higher mag here will make the RealignTo Item fail!
- If beam centering is off in View mode: in Low Dose Control,
Additional Beam Shift, select Set, center beam with the left trackball,
de-select Set. This stores an additional beam shift for just the View
mode.
- Make sure a 30 eV slit is inserted for this mode.
- Low Dose Control: de-select "continuous update of mag & beam".
Camera settings for all modes
- Camera & Macro Controls, Setup:
Low Dose mode
|
binning
|
size
|
Processing
|
exposure time
|
Dose fractionation
|
View
|
4
|
Full
|
Gain Normalized
|
0.1
|
No
|
Focus
|
4
|
Wide Half
|
Gain Normalized |
0.1
|
No
|
Trial
|
4
|
Full
|
Gain Normalized |
0.1
|
No
|
Record
|
1
|
Full
|
Dark Subtracted
|
TBD
|
Yes, Save Frames!
|
Preview
|
4
|
Full
|
Gain Normalized |
0.1
|
No
|
Camera & Macro Controls, Setup, Record mode: check that Dose
Fractionation Mode and Save Frames are selected only for Record mode,
make sure you select:
- Superresolution.
- Binning 1.
- Bin counting frames by 2 in hardware.
- Processing, Dark Subtracted.
- Dose Fractionation mode.
- Align frames.
- Save frames.
- Set exposure time for Record to get the reuired dose (use update dose button).
- Make the frame time such that it gives ~10 frames.
- We align frames on-the-fly with the P6000 GPU in the K3.
- Check GPU on-the-fly frame alignment parameters:
- We save frames as small as
possible, meaning Dark Subtracted, 8 bit and and zipped.
- Set File Options so we save frames as small as possible:
Check check!
- Camera & Macro Controls: take a View, Trial, Focus and Preview image, twice in this order.
- If the beam is off in Focus/Trial/Record/Preview: center it in any of these modes with Direct Alfgnment/Tomo Beam Shift.
- If
the beam is off in View: Low Dose Control, Additional Beam Shift,
select Set, center beam with the left trackball, de-select Set.