Autofocus Calibration with Beam Tilt Node¶

Three functions have to be performed in Beam Tilt node to perform defocus, stigmation and
z-height corrections used in autofocus procedures. Defocus and stigmator calibration are
required for defocus/stigmation correction. These plus a record of eucentric focus value are
required for the z-height correction to eucentric height.

Beam Tilt Defocus/Stigmator Calibration and Eucentric Focus Recording Need for the Example MSI:

<filename>Preset</filename>	<filename>magnification</filename>
hl	5000
fa	50000

Defocus Calibration¶

• Leginon/Presets Managaer> send the preset you want to make the calibration "To Scope"

• Leginon/Beam Tilt/Toolbar/Setting> check and make sure that it does NOT overwrite presets

• Leginon/Beam Tilt/Toolbar> select Defocus as calibration type

• Leginon/Beam Tilt/Toolbar> open "Parameter Settings" window to set parameters as follows:
  at 50,000x: defocus1 = -2e-06, defocus2 = -4e-06 and beam tilt angle=0.01
  at 5,000x: defocus1 = -2e-05, defocus2 = -4e-05 and beam tilt angle=0.01

• Leginon/Beam Tilt/Toolbar> click Calibrate.

• Occasionally the beam may be partially blocked by the objective aperture and
  produce a dark field image upon beam tilts. Reducing the beam tilt angle, using a
  larger objective aperture, and/or centering the objective aperture accurately are the
  possible solutions.

Beam Tilt Stigmator calibration¶

The Beam Tilt Stigmator calibration is used by Leginon to correct astigmatism. This
calibration functions well if the microscope has been well aligned first. Stigmation
correction at low mag such as 5000x is not necessary practically but the calibration is
still required for the equation used in autofocusing.

• Leginon/Presets Managaer> if hasn't done so, send the preset you want to make
  the calibration "To Scope"

• Leginon/Beam Tilt/Toolbar> select Stigmators for calibration.

• Leginon/Beam Tilt/Toolbar> open "Parameter Settings" window to set parameters
  as follows:
  at 50,000x: beam tilt angle= 0.01 and delta stig.= 0.2
  at 5,000x: beam tilt angle= 0.01 and delta stig.= 0.2

• Leginon/Beam Tilt/Toolbar> click Calibrate.

• Occasionally the beam may be partially blocked by the objective aperture and
  produce a dark field image upon beam tilts. Reducing the beam tilt angle, using a
  larger objective aperture, and/or centering the objective aperture accurately are the
  possible solutions.

Checking the Calibrations¶

Once both Defocus and Stigmation are calibrated against beam-tilt induced image shift,
the two can be measured using the Measure Function in the Toolbar.

• Leginon/Beam Tilt/Toolbar> click Measure to open measurement submenu.

• Leginon/Beam Tilt/Measure> click Measure (to test the beam tilt defocus and stigmation calibration).

• Leginon/Beam Tilt/Measure> click Correct Defocus to make the correction according to the measurement.

• Leginon/Beam Tilt/Measure> click Reset Defocus and exit the sub-window.

• Leginon/Presets Managaer> send the fc preset "To Scope"

• Leginon/Focus/Settings> select fc as the preset used for the acquisition.

• Leginon/Focus/Toolbar> start Manual Focusing.
  Power Spectrum of the image at defocus specified by "fc" preset should appear on
  the screen.

• Leginon/Focus/Manual Focus> set 0 defocus to scope by selecting the movetype
  to "defocus" and the number entry in meter to "0" and then click "Set
  Instrument".
  The Thone ring should disappear as the defocus is changed to zero measured by Beam
  Tilt node. If not, recalibration of defocus may be needed.

• Leginon/Focus/ToolbarManual Focus> stop Manual Focusing by clicking the "stop" button.

• Leginon/Beam Tilt/Toolbar> click Measure to open measurement sub-menu.

• Leginon/Beam Tilt/Measure> click Correct Stigmator to make the correction
  according to the measurement.

• Leginon/Focus/Toolbar> start Manual Focusing and check to see if the
  correction makes bad stigmation better.
  If not, redo stigmation correction.

Trouble Shooting:

• The microscope must be well aligned. The most critical are beam tilt pivot point and rotation center, both should be aligned at eucentric focus. Please refer to Microscope Set-up to align the microscope well.

• Chose a beam tilt and defocus change that will give a 10-20% shift in the image.

• The beam should cover the entire CCD imaging area.

• Have an easily recognized object at the center of the image for easy correlation. Avoid uneven areas.

• Watch the images movement. There should not be any contrast reversal or appearance of the edge of the beam in any image acquired. Contrast reversal is caused by misaligned objective aperture or by too large a beam tilt angle so that part of the beam is blocked by the aperture when tilted.

• Check the correlation image and peak location to find out if the image contrast is too low for good correlations.

• The default values to correct calibration the stagmators are recommended. If these values do not produce good stigmation calibration results and if the microscope has been well aligned, then progressively and slowly change the Stigmator Tilt and Stigmator Delta values until the images cross correlations result in measurable shifts between steps of the calibration.

Eucentric Focus Storage and Retrieval¶

Encentric Focus From/To Scope (From/To Scope icons) do what they indicate. The "from
Scope" function simply records the current absolute focus value at the scope.
Therefore,

• Scope> eucenter the grid and reset defocus at U-centric height at the required high tension and at a high mag where focusing is easier.

• Leginon/Presets Manager> select the preset that will perform z-focusing (i.e., hl). temporarily change its defocus to 0.0

• Leginon/Presets Manager> sent the preset "To Scope"

• Leginon/Beam Tilt> left-click the Encentric Focus To Scope icon to save the focus value.

• Leginon/Presets Manager> return the defocus value back to its original.

Rotation Center Storage and Retrieval¶

Rotation Center From/To Scope (From/To Scope icons) do what they indicate. The "from Scope" function simply records the current beam tilt value at the scope. Therefore,

• Scope> Align the rotation center at a magnification and high tension where the value will be used.

• Leginon/Beam Tilt> left-click the Rotation Center To Scope icon to save the beam tilt value.

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