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.

<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 subwindow.

• 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 submenu.

• Leginon/Beam Tilt/Measure> click Correct Stigamator 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 <link
  linkend="microscope_setup">Microscope Set-up</link> 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 a easily reconized object at the center of the image for easy correlation.
  Avoid uneven area.

• Watch the images movement. There should not be any contrast reversal or apearance
  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 measureable
  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.