Leginon

All matrix calibrations are dependent on the high tension, so they will have to be done again (including defocus and stig).

If you are using a modeled stage calibration, you will have to at least get a new "mag only" calibration for any mags you are using at the new high tension. The actual model is not dependend on high tension, just the "mag only" part.

The eucentric focus (used for doing Z focusing) is also dependent on high tension.

Pixel size calibrations depend on high tension, but I think we ignore that dependency in a few places where it is used.

Once you think you have calibrated everything, a good check to be sure you are calibrated at the current high tension is to look in the presets manager. It will list when the most recent calibrations were done for each preset at the current high tension.

I will have one of the single particle experts (not me) reply about choosing the right high tension.

Jim

The voltage you choose depends on the specimen. We like 120KeV for "smaller" things like GroEL because we get better contrast and still achieve high resolution. For things like big viruses, you might want to use 200KeV so that you can penetrate the thicker ice where the viruses are. Even so, sometimes 120KeV can be beneficial for generating contrast. [/quote]

Hi,

I am also considering data collection using different voltages. So far, we have used only 300 kV, but 200 kV or even 120 kV should give better contrast for small particles. On a tecnai microscope, the alignment has to be done for each voltage and can be stored separately. What about Leginon? Will Leginon store calibrations for different voltages separately in the database or is it necessary to re-calibrate everything, whenever the high voltage is changed? What is the easiest way to work with different voltages (e.g. creating individual sessions/applications/presets)?

Thorsten

Leginon will store different calibrations for different voltages, but this is transparent to the user in most cases. When you do a new calibration, the voltage is read from the microscope, and then the calibration is stored in the database with the associated voltage. When Leginon needs to use a calibration, it will again read the current voltage from the microscope and then query the database using this voltage. Therefore, you do not have to recalibrate every time you change voltage, but you do have to make sure you do the calibration the first time you use a new voltage.

By "calibrations", I am only refering to the following, which have an associated voltage recorded in the database:

-Matrix calibrations for movement and focusign (stage, image, beam shift, beam tilt)

- Magnification component of the modeled stage calibration (not the actual model)

- Camera sensitivity recorded during dose calibration

- Eucentric focus

- Rotation center

Currently, presets are not associated with a voltage (although they probably should be). This is something to remember when you import presets from a previous session. If the previous session was done at a different voltage, then those presets may look very strange. For now, Leginon still allows you to import presets from any session, so it is up to you to determine if those presets are going to work at the current voltage. Mainly, you may have to make significant adjustments to intensity, beam shift, and image shift. You would probably have to adjust these things a little anyway, even if you imported presets that were at the same voltage.

To keep things organized, it is probably best to use only one voltage during a session, although there is nothing stopping you from changing it halfway through.