20 November 2010, R. Ubic
15
Tomography
Insert the holder and bring up the beam at 3000x. Make sure the beam is spread out but
don't go all the way to fully overfocus with CL3.
Acquire a trial image with SerialEM. If what you see looks contrast-rich and has many
features, start 'eucentric - rough' from the task menu.
Once this completes, locate your ROI and set the magnification and brightness (you can
move the stage and set the brightness all through SerialEM).
Once you have an ROI for tomography run 'eucentric - fine' from the task menu.
Walk up the stage to 53 degrees using 'walk up' from the task menu. This starting angle
may be higher if you use a High Tilt Retainer and no in-gap OL aperture in the beam
path.
From the 'tilt series' menu choose 'Setup/start' to evaluate your tomography parameters.
Make sure that the angles are properly specified and pick your basic increment (1.5 or 2
is perfectly fine, or if you want to get more detail in the tomogram you can pick 1
degrees). Pay attention to the mag and the 'at start and when needed track at
magnification'. Rule of thumb here is the latter should probably be ~1/3rd of the imaging
magnification.
Keep the beam intensity for the moment constant.
Most biological samples do fine with a focus of -0.35 micron, so do most materials
samples. For cryo you need more, say 3-6 microns. If the ROI has features that only
show with a lot of defocus, you can add this as an offset to the autofocus (remember
that underfocus is negative!).
For initial action you probably should have a trial image in focus before you bring up this
window, so 'align to image now in Buffer A' should be on.
Leave tracking unchanged for the moment.
Hit 'GO' and sit back. Remember there are 'End' and 'STOP' on the camera and macro
controls panel that will halt the tilt series acquisition.
Once the series is done, terminate it from the 'Tilt Series' menu and close the file and do
not leave 90° rotated copies in various buffers.
You can run up the tomogram in etomo.
