Electron Tomography - Taking a tilt series using EMMEMU
[MODES]
[TEM LOW DOSE]
Enable joystick Z (for tilt series):
[COMPUSTAGE]
[COMPUCTRL]
[JOYSTICK Z]
§ Ready
Without inserting a holder do flatfields
Make sure that the vacuum < 17
Bean on by rotating filament knob slowly.
flatfields / background subtraction:
confirm beam is centered and spread the beam to get meter=2.06 s
raise screen; cover view field with mat
Repeat process below for each camera format used:
Format …
> select desired format (typically,2k x 2k)
OK
Flatfields …
Exposure (uncorrected)
adjust exposure to desired mean (typically 2000)
Acquisition:
Exposures: 4 (min) – 8 (max)
all boxes unchecked
Create Flatfield
Check and Save:
Exposure (corrected)
should show featureless grainy image
Store Flatfield
Close
Beam off
Load a sample onto a high tilt holder and insert it.
Wait until vacuum gets down to 16
Bean on
[SEARCH]
Format …
> 0: 2048X2048, 1X, …
OK
take test exposure to update format
adjust exposure time for appropriate mean.
align [SEARCH] and [EXPOSURE] modes
for [EXPOSURE] mode:
D (diffraction mode on)
use eyepiece focus needle for correlation
[SEARCH]
> Spot size: spot 3
use highest Spot for which you can see detail
find a unique “landmark” near region of interest and adjust to focus
[EXPOSURE]
> Spot size: spot 6
D (diffraction mode on)
Adjust to focus and center on [SEARCH] area using multi-function knobs
[SEARCH]
adjust to focus
Exposure to test
[RESET DEFOCUS]
higher mag = lower defocus
-4um for 25,000X
-10um for 12,000X
to check precise defocus
Power
Power [>] Fit PCTF
increase defocus to fit rings to zeros
Apply
Adjust defocus as necessary
Exposure to test
Format …
> 1: 1024X1024, 2X, …
OK
Set exposure to 100 ms and take test exposure to update format
adjust exposure time for appropriate mean if necessary.
Keep the screen up
Positions …
Show positions
SCANNING, EXPOSURE, TRAKING tab, check magnification
Calibration …
Update TEM
Calibrate Gonio X/Y
Calibrate tilt axis
Calibrate communication
Calibrate image shift
Calibrate beam shift
follow instructions
Close
Show positions
SCANNING tab
Beam shift x:0
Beam shift y:0
Image shift x: 0
Image shift y: 0
Check the spot size
Apply
Exposure, tracking, and Search tab
Check sopt size
Apply and close
> SCANNING
Goto position
Update Position
> ALL
Copy position to
√ Lock positions
Show positions
SCANNING tab
Defocus: 0
Offset defocus: 0
EXPOSURE tab
Format:
> 0: 2048X2048, 1X, …
Exp. time (ms): 50-100% of standard exposure time
check mean, modify as needed
Defocus: 0
Offset defocus: 0
FOCUS tab
Defocus: -5000
Offset defocus: -5000
TRACKING tab (for tilt series)
Exp. time (ms) adjust for same mean as EXPOSURE mode
Defocus: -5000
Offset defocus: -5000
SEARCH tab
Format:
> 1: 1024X1024, 2X, …
Magnification: ~½ mag for EXPOSURE mode
Exp. time (ms): lowest possible exposure time to yield detail
Defocus: -20000
Offset defocus: -20000
Apply and Close
Close
test beam centering:
use desired magnification
focus beam and center with shift x/y knobs
raise and lower screen to check for beam shift
repeat calibrations if necessary
move to an area close to your object
eccentricity correction
[EXPOSURE]
§ D (diffraction mode on)
y coordinate must be within 100 of origin
correct for eccentricity near region of interest
same y coord as region of interest
[COMPUSTAGE]
[A-WOBBLER] on
adjust Z so field doesn’t shift
[A-WOBBLER] off
[EXPOSURE]
Goniometer Tilt
tilt to ±70°
adjust Z so area stays in field for all tilt angles
[SEARCH]
Exposure
check focus and eccentricity. compare with previous exposure
[EXPOSURE]
· D (diffraction mode on)
relocate to region of interest
v Goniometer Tilt
tilt to -70° or 70°
raise screen and cover view screen with the mat
[SEARCH]
Positions …
Center detail
Update Position
> ALL
Copy position to
Areas
tracking should be outside of exposure area along tilt axis
search area should include exposure area
Tilt series
change directory and file name as needed
Start angle: -70 or as needed
End angle: 70 or as needed
√ Exposure every
√ Tracking every
√ Search every
√ Focus every (if not cryo)
√ Angles by Saxton scheme with 0° increment 2 or 3 deg
√ Fully automatic tracking
√ Display focusing steps
√ Display tracking steps
START
¤ Add entry before: 0
Entry to add:
Position:
¤ Exposure
Angle: current angle
Do edit
¤ Search
Angle: current angle
Do edit
adjust meter to 2.06
cover view field w/ mat
lift screen
Start series
to manually recenter during tilt series acquisition
press spacebar at XCF screen
Continue semi-automatic
Center detail
recenter using tracking image
Continue full-automatic
Convert .dat format to .tif: and generate save_txt file
user date
make sure spe ang. spe dir. mean are checked.
close
Open series
¤ EM-FORMAT
CONTINUE >>
…
Save series
Same as saving regular images