How to do Calibration
Added by Yen-Chywan Liaw over 17 years ago
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I am trying to do calibration application. I follow the Manual creat preset (gr, sq, hl, fc, fa, en, ef) And input most of values copy from manual. Now I have questions
1. what exactly do I need to do to adjust preset at microscope? (there are only beam shift and Intensity I did not input, but I thought Beam shift is going to calibrate later????)
2. If I go to next step without do any adjustment, to assign pixel size. I can not see any magnification shown on table. Why??? And there is a Error indicator shown next to a node in node selector.
3. If I click Toscope at preset manager and go to pixel size. This time I can see 39 different magnification. But I still got error indicator next to node in node selector. I input pixel size for most of magnification. But they seem not saving. They disppear when I next login.
4. what kind of grid with what sample on it to use. especially at dark reference and bright reference acquisition?
Yen-Chywan
Replies (3)
- Added by Anchi Cheng over 17 years ago
Regarding preset adjustment at microscope:
Presets are microscope/camera configuration for acquiring images. Therefore, apart from setting the magnification, camera configuration etc. you certainly want to make sure that the electron beam are more or less centerred on the CCD camera when your preset is sent to the microscope to take images. That is where beam shift comes in. Similarly, you need to decide on the size of the beam which affects the intensity of the beam and therefore the correct exposure time for your presets.
The section on "Creating Presets the First Time" in the chapter for "Calibration" shows the most straight forward way to create presets commonly used in MSI applications. To be more specific, you adjust the magnification knob on the scope to the value you want for the preset and then adjust the beam shift to center on the CCD and expand it to the appropriate size. Then you go the next steps to save them "From Scope" under the preset name. I will add these specific instruction in the future manual.
Regarding no magnifications in the pixel calibration node the first time it is opened----
This might be because the node had no information on what instrument was used, yet. Is the error message "Preset is unknown and preset overide is off"? When you sent a preset to the scope, an event was sent to the "Pixel Size" node that contains the instrument information. Therefore, the magnification table shows up.
The error/warning indicator does not clear evn if the problem is removed and the node continue its function. They can only be cleared by deleting the logging message in the logger window. This is done on purpose but does cause confusion. When Leginon is run automatically and the user steps away from the computer, the warning indicator need to stay even if the node has moved on. This way the user can check and know it has occurred when he/she gets back.
Regarding the values not been saved----
This seems strange. The edited values should be saved to the database and the table in the "Pixel Size Calibration" window updated when the "Save" button is clicked in the "Edit Pixel Size" window (Not just closing the window). Its behavior is the same as any other entries in Leginon. Do you have the same problem in other places such as in "Presets Manager"?
Regarding what grid to be used for calibraionts----
Any grid with good contrast and features for easy cross-correlation at various magnification will do. An example would be a 300-400 mesh em grid that has negatively stained protein on either quantifoil or continuous carbon support. As for dark and bright reference, you need to have no scattering material in the view of the CCD. In other words, it can be a grid square with on support film from the above-mentioned grid, or you can have no grid at all. Again, this is something that was not spelled out in the manual. I will add that.
Anchi
Re: How to do Calibration - Added by Anonymous about 15 years ago
Related to this topic:
I've been working on the calibrations on multiple sessions over the past week, and I noticed that whenever I resumed the calibration process, I had to retake the reference images (dark, bright, raw, corrected) because Leginon couldn't find them. It did remember the presets, though.
Am I supposed to retake the reference images each session or are they stored somewhere in the database?
Thanks for the help!
Re: How to do Calibration - Added by Jim Pulokas almost 15 years ago
You should not have to redo reference each time. Does this only happen when you create a new session, or just when you restart Leginon with the same session? To help us figure it out, can you run a quick test to make sure the problem is still happening, then make a dump of your username, hostname, and database name to a file and then send us the file. You can dump the database to a file like this:
mysqldump -h dbhost -u dbuser -p dbname > dump.sqlreplacing all the specifics of your database, for example, your dbname may be dbemdata if you followed the install manual exactly. If you do not use a password, you do not need the -p. That will dump to a file dump.sql which you can attach to an email. We should have some time to look it over early next week.