Leginon

h1. 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>

h2. 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)

h2. 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)

h2. 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.

h2. 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.

h2. 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(or LM)/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(or LM)/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.

h2. 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.

h2. 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.

h2. 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.

h2. 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.