Troubles with Imaging - Troubleshooting¶

• <link linkend="imageno">Empty Image</link>

• <link linkend="imageartifact">Image or FFT contains artifacts</link>

• <link linkend="cor_not_read">Reference image from other users not readable</link>

• <link linkend="beamshifttime">Beam shifts away from the imaging area over
  time</link>

• <link linkend="beamshifttime">Beam shifts away from the imaging area after a large
  defocus correction</link>

• <link linkend="beamshiftimage">Beam shifts away from the imaging area upon image shift
  targeting</link>

• <link linkend="beamshiftstage">Beam shifts away from the imaging area upon stage shift
  targeting in LM mode</link>

• <link linkend="apertureoff">Objective aperture is off-centered in LM when it is centered
  in HM</link>

• <link linkend="specimenoverdose_gatan">The specimen appears over-dosed in the high
  magnification images (while using a Gatan CCD)</link>

• <link linkend="specimenoverdose_gatan">Bright/Dark correction has no effect on the image
  (while using a Gatan CCD)</link>

• <link linkend="badinput">Acquired image configuration is not what was
  inputted</link>

• <link linkend="melt_beam_imprint">Exposure image has a circular focus
  imprint</link>

• <link linkend="melt_beam_imprint">Image acquired at the wrong stage
  position</link>

Image is acquired but contains no real information¶

Commonly Why:

• Main screen at the scope is down

• Camera is not inserted (Gatan camera)

• Digital Micrograph program is not running (Gatan camera)

• Shutter switch box is set to CM, i.e., to collect film data only (Tietz
  camera)

Image is acquired but contains artifacts either in image or
in Fourier Transform¶

Commonly Why:

• Beam is partially covering the CCD.

• Scope viewing window is not covered and room light is on

• Dark/Bright images are not available or need to be reaquire.

• Bias level and quadrant correction may need to be reset (Digital Micrograph and
  Gatan camera)

Can not read reference images that were acquired by other
users¶

Commonly Why: You don't have permission to read their image files

Solution:

• Have the person change the group or even world permission for reading the data
  directory and the image files in it.

The person is not around when this happens:

Acquire your own reference images at the camera setting that needs the dark and bright
images. However, Leginon always look for the most recent calibration in the database by
anyone. Every user will have to do this everytime if the permission problem is not
solved.

Beam shifts away from imaging area over time¶

Commonly Why:

• Residual lens hysteresis if you haven't allowed the scope to warm up for HM mode
  after acquiring LM atlas.

• Focus correction has taken the absolute focus point far away from U-centric
  focus.

<title>Solutions:</title>

• Wait until the optics is stabilized

• Activate "Publish and wait for the reference target" in Exposure Node. This will
  enable "Fix Beam" node to search for the beam in the desired range and shift the beam
  to the best result. A reference target, normally an empty square, need to be selected
  on the grid to use this function. See description in the node description chapter for
  usage.

• Pause MSI and correct for residual beam shift

• Pause before next hole or square by clicking Hole/Toolbar>Pause button
  (Pause icon) or Square/Toolbar>Pause button (Pause icon), respectively. Which
  one to pause depends on what is the next target.

• Leginon/Presets Manager> When the acquisition sequence is stopped, send
  the preset that has beam shift problem "To Scope" (ToScope icon).

• Leginon/Navigation/Toolbar> Choose move type to be "Beam Shift" and make
  sure "Use Camera Configuration" is unchecked in settings.

• Leginon/Navigation> "Navigate"*(navigate icon) to center the beam. If
  "Navigate" does not bring the beam in, manually shift the beam at the
  microscope. Do not forget to lift the screen and cover the viewing area after
  done.

• Leginon/Presets Manager> save the new beam shift by retrieving it "From
  Scope" (From Scope icon).

• "Continue" data collection on next hole or square target by clicking
  Hole/Toolbar> "Continue" button or Square/Toolbar> "Continue" button,
  respectively.

*"Navigate" means selecting the navigate tool on the top right of image
display and then single-left-clicking the location of the new center that is
translated by the given TEM Parameter.

• Reset the defocus=0 point back to U-centric focus.

• Leginon reset defocus after each defocus correction. Therefore, the point where
  the user defines as defocus=0 may drift away from U-centric focus over time or after a
  very large correction before it is reset to U-centric focus by z focus
  correction.

• Pause before next hole or square by clicking Hole/Toolbar>Pause button
  (Pause icon) or Square/Toolbar>Pause button (Pause icon), respectively. Which
  one to pause depends on what is the next target.

• Leginon/Presets Manager> Send "hl" preset "To Scope" (ToScope
  icon).

• Leginon/Z Focus/Toolbar> Send stored U-centric focus to scope.

• Leginon/Z Focus/Toolbar> Reset defocus to 0

• Scope> U-center the stage if it is very out of focus.

• Leginon/Presets Manager> Send the preset that showed bad beam shift "To
  Scope"

• Scope and Leginon/Presets Manager> Check the beam shift, make
  correction, and then retrieve the correct value "From Scope"

• Leginon/Drift Manager> Declare Drift (declare drift icon) to force
  correction at next possible point in the process.

• "Continue" data collection on next hole or square target by clicking
  Hole/Toolbar> "Continue" button or Square/Toolbar> "Continue" button,
  respectively.

Beam shifts away from imaging area only when targeting
move type is image shift¶

Commonly Why:

• Image/Beam Shift calibration on the microscope is not optimal

• Defocus is very far from eucentric focus

<title>Solutions:</title>

• Bad Image/Beam calibration

Follow instruction for performing the calibration for FEI Tecnai machines under
Alignments/Image HM-TEM/Image-Beam calibration

• Pause before next hole or square by clicking Hole/Toolbar>Pause button
  (Pause icon) or Square/Toolbar>Pause button (Pause icon), respectively. Which
  one to pause depends on what is the next target. If in queuing mode, pause at the
  node that process the queue.

• Scope> move to an unimportant area or pull the holder out enough to allow
  the beam to go through.

• Scope> reset defocus to eucentric height.

• Scope> Follow instruction for performing the calibration for FEI Tecnai
  machines under Alignments/Image HM-TEM/Image-Beam calibration.

• Leginon/Presets Manager> Cycle the presets a few times. The calibration
  takes the scope to conditions outside the presets, and can cause strong hysteresis
  if this step is not done.

• Leginon> Follow the procedures for confirming and saving good image shift
  at various presets.

• "Continue" data collection on next hole or square target by clicking
  Hole/Toolbar> "Continue" button or squares/Target Toolbar> "Continue"
  button, respectively.

*"Navigate" means selecting the navigate tool on the top right of image
display and then single-left-clicking the location of the new center that is
translated by the given TEM Parameter.

• Defocus far from eucentric focus

Image/Beam shift coupling worsens when the defocus is away from where it was
calibrated. If, after the above calibration, the problem remains during MSI
acquisition, additional focusing sequence should be added at lower magnification that
move the stage to close to eucentric height so that the image shift target is selected
on an image of a close-to-eucentric location.

• Leginon/Z Focus/Focus Sequence> activate both Stage_Wobble and
  Z_to_Encentric steps.

• Leginon/Hole (or Subsquare) Targeting> selection a focus target if not
  already done so when a newly acquired sq image comes in.

If the accuracy of moving stage to eucentric height by the focus sequences in "Z
Focus" is still not sufficient. Add another focus step performing the same task as
Z_to_Encentric step. Repeating beam-tilt based autofocusing often improve the accuracy
unless the calibration is off.

Still not working: You have chosen a target that requires too much image shift for an
independent image shift from beam shift. This is necessary when the lower mag targetting is
not good either because the preset image shifts are not aligned or the stage position
movement is not properly modeled.

Solution: For image shift problem see solution for "Target is consistently off in the
same direction and distance". For stage model problem, see "Modeled stage calibration" in
setup notes.

Beam shifts away from imaging area when targeting move
type is stage movement¶

Commonly Why:

• U-center defocus not set to 0 in LM mode.

• LM alignment such as rotation center is way off.

Solution: Check and correct microscopic alignment error. Preset parameters checking and
reset may be necessary after the correction.

Comments: If the problem persist, the original stage matrix calibration may have been
performed with a bad alignment. The calibration should be redone with a well-aligned scope
in LM mode.

Objective aperture appears off in "sq" preset when it is
centered in HM mode¶

Commonly Why:

• U-center defocus not set to 0 in LM mode.

• LM alignment such as image shift is way off.

Solution: Check and correct microscopic alignment error. Preset parameters checking and
reset may be necessary after the correction.

The specimen appears over-dosed in the high
magnification images or the dark correction image has a high value (while using a Gatan
CCD)¶

This issue may occur when the shutter is incorrectly configured through Digital
Micrograph (the imaging software for the Gatan CCD that resides on the TEM computer) or
simply a bad dose calibration or too long an exposure time. To issue these
corrections:

• Set the "shutter" to "Auto" on the external control box attached to the
  TEM.

• Open Digital Micrograph on the Microscope computer and go to the Camera Set-up
  page.

• Go to Configuration

• Set the Primary Shutter to Pre-specimen

• Set the Alternate Shutter to Post-specimen

• Set the Default Primary Shutter to the Normally Closed position.

The exposure image has a circular imprint of the beam
at the size of the preset used for melting the ice¶

This issue may occur when the main screen is left up during very long (such as over 30
sec) ice melting and at high HT. Leginon has a mechanism that put the screen down during ice
melting. It is caused by over-saturating the Phosphur layer on the CCD. If you see this
effect, the screen may have failed to lower. Please test it by observing the screen movement
with simulating target in any focuser node that has ice melt time set not to zero. Report
the problem back to the leginon team if you are sure it is not your scope's problem.