Focus and Z-height Setup
Added by Anonymous almost 15 years ago
Just a couple of questions regarding how Leginon goes about focusing.
A) Does Leginon store the Z-height of the microscope when eucentric is sent from the scope to the host? We've been trying to figure out how Leginon stores the Z-height and whether it does so similarly to how it stores the defocus in the Focus node. This is what we tried:
1) Set the defocus and the Z-height manually early on (before obtaining a grid atlas) in the align square; reset the defocus. The Z-height value is X microns, defocus is 0.00 microns.
2) Continue with the MSI setup until we get to the hole targeting/exposure targeting step.
3) Switch on the "manual after" options for both Z-height and Focus nodes.
4) Z-height is adjusted by Leginon (new Z-height is X + A microns, defocus still 0.00 microns). The value of A has some error in it (I think).
5) Manual focus shows up with the fa preset switched on (setting defocus to -2.00 microns). Some Thone rings are visible. Setting defocus to 0.00 microns does not remove them (since it hasn't passed through the Focus node?). Manually finding the defocus (without adjusting the Z-height) removes the Thone rings.
6) We deliberately moved the stage away from eucentric height (on the scope) to a new value (X + A+ B)--Thone rings appear. We then try sending the eucentric to the scope, but all it does is set the defocus to 0.00 microns (Z-height is still X + A + B when it should be either X + A); the rings do not disappear.
7) We find the real eucentric (X) manually, and proceed to the Focus node--it's able to find the defocus properly as seen in the manual defocus (though this could be because we set it earlier...)
The last step seems to imply that Leginon isn't storing the eucentric height like it does the defocus. Should it be storing it (that is, storing the value of X + A it found)? How important is it that the defocus is obtained in step 1 before targeting anything if it's just going to try to find it anyway?
B) What sequence should we follow in setting up the focusing? The MSI quick start up seems different than the one in the longer explanation (for instance, it doesn't go through target simulation) and it's unclear of when the user should set the eucentric focus on the machine.
C) Is there a way for Leginon to correct astigmatism? In the calibration application, there seemed to be an option for having Leginon correct it (it was one of the testing steps) but in the actual operation, this option doesn't seem to show up.
Thank you for the help!
Replies (2)
Re: Focus and Z-height Setup - Added by Anchi Cheng almost 15 years ago
Carlos,
Sorry for ignoring you for a few days. We have other users need more urgent help.
(A)---------------------------
First, Leginon does not store eucentric height. While eucentric height is a fixed value relative to the column, different grid weighs down on the goniometer differently. Therefore the stage Z value is not a constant for various grids and stages when the eucentric condition is met. We store eucentric focus. This is a current applied to the objective lens which we can return to reasonably well. By the way, we usually use the encetric condition where the stage tilt creates minimal movement of the sample observed. You can use the eucentric focus value stored in the microscope (recalled by pushing the eucentric focus button on the microscope control pad) if you are not going to do RCT or tomography. For these two purpose, the former stage tilt definition is more where you want.
Therefore, your observation in (6) is correct. Send Eucentric Focus to scope does not make a grid that is off eucentric height to move to eucentric height. Sending it, however, make it possible to measure the displacement of the stage from eucentric height (relative to the scope column).
About saving Z-height at X+A at the end of Z_Focus node, well, it kind of did. When Leginon proceed to acquire images of the holes that were chosen on the same square, if you look carefully, the stage Z value will be whatever value you left at when you close the manual focus, or more correctly, whatever Z_Focus node make it to be.
What you do in step one is done so that you have a consistent starting point from session to session. The presets are set to the scope according to defocus relative to where you reset to zero. If the "zero" is very different from session to session, it will be difficult for you to import previous presets and trust them be aligned and at similar scale (remember that heavy defocus can distort the observed magnification).
(B)--------------
Follow Quick Start up sequence and use the long explanation for details on how to do a particular step. If you have to repeat testing autofocus, the simulated target Alternative procedure is quite handy. Saving eucentric focus from scope in Quick Start used to be something requires little explanation. Therefore, I didn't explain it more....
(C)---------------
There is a calibration in Beam_Tilt calibrator node for astigmation. If you watch the correlation peak move during the calibration, you would notice that the movements are very small when the astigmatism is applied at various steps even though the change is obvious in the image characteristics. We found that the correction obtained from such algorithm is not accurate because the small correlation peak movement at moderate astigmatism. The measurement is also too sensitive to factors such as drift to be used reliabily with ice sample. Therefore we did not emphasize it in the procedure. You can turn it on if you like. It is available for any beam-tilt based focus step. You can open up the focus sequence settings dialog window in Focus node, select one of these focus step. It is a checkbox at the bottom. You can specify the range of defocus that you trust the measurement and want to do the correction since the accuracy of the measurement is defocus (percentage of image shift at a given astigmatism(delta defocus)) dependent.
I am glad that you start to study Leginon's behavior. I hope you get good data from it.
Anchi
Re: Focus and Z-height Setup - Added by Anonymous over 14 years ago
Hi Anchi,
Thank you! Your explanation of the focus setup was very helpful. Sorry I took so long to get back. I still have some questions about the autofocusing that I would be very appreciative if you answered, though:
A) It's not entirely clear to me why the defocus on the fa and fc preset have to be set to -2.00 um. The autofocusing wasn't focusing correctly for a while and was consistently off by 2 or more microns, but when I changed the focus presets to have a 0 defocus, it behaved much more consistently (and accurately) for some reason. If the issue is there not being enough signal for the program to correlate, then why not use a larger defocus (e.g. -4.00 um)? I tried this and I think it actually made it worse, but I'm not sure why. I'm sticking to 0 um for now, but I'd like to know if there is a compelling reason to use -2 um.
B) I noticed in the set up that when testing the Z-height auto-focusing, you have to choose a focus target in the ice (preferably with some visible debris on it). I used to forgo doing this and I think it's one of the reasons why the defocus autofocus behaved inconsistently before. Now that I specify the focus on the carbon, it's been focusing more accurately and precisely (though it's still 0.5-1.0 nm off now and then). It makes sense that leginon would want to use the carbon to focus on, which is why I am confused about why the default insists on choosing to focus in the ice of a "good hole/any hole" (in the hole auto-targetting setup). I've tried specifying an offset (like in the exposure targeting setup), but it still chooses to focus on the middle of a hole and not on the carbon. Have you guys encountered this problem before?
Thank you very much for all of your help and counsel!
~Carlos