Leginon

what is your aperture size? I assume you are talking about objective aperture, right? What is the imaging area size in meters at your sq mag, and what is your mag? What is the em grid square opening size? What much of sq image does the aperture cover? If you have example images, please post it.

My guess is that some alignment is bad, but it will help determine if I have the full information.
Moving stage should not change the image shift alignment unless the alignment is aweful.

Anchi

Hi Anchi,

Thanks for the reply--I'm waiting to hear back from someone who knows those values; the aperture seemed to cover only about 1/4 - 1/5 of the grid square area (so not too much), but that is as far as I personally know.

I will try to get a picture to you so you can see what I mean. What is the easiest way of extracting an image from the database? I can find it fine through the webviewer, but I haven't played around too much with it. I can find some pictures by rooting around the raw-data folder if need be, but I thought I should learn now. I would be happy to read the manual, but I can't find info on how to use it anywhere there... I'll figure it out, don't worry! <img src="{SMILIES_PATH}/icon_biggrin.gif" alt=":D" title="Very Happy" />

Cheers,

~Carlos

Carlos,

Best is to go through the web viewer. The information tool on top of the image will give you the image path, name and all other information. Plus, you can just take a screen shot of the page because it will have scale bar, magnification, pixel size that I need to know anyway.

Hi Anchi,

I sent you the images showing the field of view of the ccd camera once the aperture is in (at low mag). This is approximately what we see whenever we try to navigate and the aperture is in.

~Carlos

Carlos,

Your images show that the apperture covers more than 2/3 of the imaging area. This certainly will not work. The magnification we suggested in the documentation works for us for our specific aperture size, pixel-size, and post-magnification of the camera. No two microscope/camera pair is exactly the same. To make the area free of blocking by the aperture in sq moder, one solution is to go to higher mag for your sq preset. You may not be able to cover the entire grid square in the area shown within the confinement of the aperture but, as you've discovered, you have little choice if that is the aperture needed for your sample.

Some people go as high as using the lowest M mode mag to do this instead of LM mode with great success. There are two advantages in using M mode.

1. The objective aperture will not show in the image because it now applies to the Fourier space to limit the resolution.
2. If you skip gr preset in cycling, you will always have objective lens on. Therefore, your image shift alignment should be more stable which translates to more accurate targeting of the holes on the square images.

Of course, at higher mag, you may have to pick a few positions on one grid square on your grid atlas. It will not upset Leginon when you do so. Therefore, no harm done, just takes a bit longer.

Now, you seem to indicate that your stage movement is not accurate enough even to reach the similar area when a position is recalled. This would be something you need to get FEI engineers involved. A properly functional stage should take you back, when called for within 2 um, I believe. You can try that on FEI GUI by saving a position and then try to reach the same position from different part of the grid by telling it to go to the saved position.

If your scope passed that test and when it is idle, the number shown on the GUI does not move by itself by a large number, your calibration might be really bad. I will need to see your calibration matrix. Start the calibration application, send your sq preset to the scope, and go to Matrix and find the tool on top that says edit. When you click on that, it will pop up a window and show you the current calibration. Post that number here, along with the pixel size. Do the same with gr preset. In addition, send me the images of the Goniometer Model graph from http://yourhost/dbem/admin.php under Goniometer. You might have over-fitted it.

Anchi

Hi Anchi,

I think the stage position calibrations are ok, but I can check them. When the aperture is out, the movement has been fairly accurate (~1-5% error), but I can get the calibration data to you the next time I have time on the microscope if you think it will help.

I will definitely try using a different magnification for sq, though. What things would I need to change for the preset besides the magnification? Should the image shift, binning, beam coverage, exposure, and spot size be the same if I'm changing it to an M magnification? I'll go through the other calibrations since those will also certainly change.

Thanks for the help so far! I'll let you know how things work out.

~Carlos

Carlos,

If you feel that the calibration is not the problem, you don't have to send me those data.

Just use your common sense in determining the binning/exposure time to get a usable image. Again, make sure the beam covers most of the image. In low mag M mode, you might see some aperture-like black area, too. It comes from some other restriction in beam path in the scope column. Just try an image shift navigation at that mag after image shift calibration to see if the aperture effect will cause any correlation problem. If so, go to higher mag. We normally keep the spot size the same because there is enough range to adjust exposure time to get a reasonable image intensity. There is a small hysteresis effect between spots and can make preset changing slower.

Anchi