Leginon

h1. Troubles with Targeting

* <link linkend="atlassmall">Grid Atlas is too small to see the details</link>

* <link linkend="atlasbad">Grid Atlas is not well patched</link>

* <link linkend="atlassmall">Grid Atlas displayed in Leginon and the Viewers on the web

are not the samei</link>

* <link linkend="atlassamelabel">Grid Atlas displays incorrect current

position</link>

* <link linkend="toffinsamedir">Targeting is consistently off in the same direction and

distance</link>

* <link linkend="toffindiffdir">Targeting is consistently off in opposite direction from

where you pick</link>

* <link linkend="toffinconsist">Targeting is off in an inconsistant way or goes off again

in the next run after correction</link>

* <link linkend="toffawayfromc">Targeting is off more for targets away from the center of

the image</link>

* <link linkend="hlfindfail">Auto hole finder fails to find holes with good ice after set

up</link>

* <link linkend="no_broken">There is no broken square for reference target</link>

h2. Grid mosaic is too small to see the details

<title>Why:</title>

* The image scale for creating mosaic is set too small.

<title>Solution:</title>

*  Leginon/Square Targeting/Mosaic Settings> Change "Scale Image" to a larger

size

*  Leginon/Square Targeting/Mosaic Settings> Click "Apply" and then "Create" the

mosaic atlas.

h2. Grid mosaic is not patched together well

<title>Commonly Why:</title>

* LM mode U-center defocus not set to 0.

* LM mode rotation center is off.

* Bad stage position matrix calibration

<title>Solutions:</title>

*  Correct the alignemnt error or redo <link linkend="mcal_st_pos">stage position

matrix calibration</link>

*  Acquire a new atlas

*  Leginon/Grid Targeting/Setting> Enter a new label

*  Leginon/Grid Targeting> Click "Calculate Atlas"

*  Leginon/Grid Targeting> Click "Submit Targets" (play icon) to begin

acquiring the atlas.

*  Leginon/Square Targeting> Follow the atlas progress in the

display.

h2. Grid mosaic display in Leginon is different from that of the

Web Viewer

<title>Why:</title>

* The goniometer does not reach the intended position due to ,most commonly, a pole

touch error that temporaily disables the goniometer.

* The calibration used to move to the target in "Grid" node is not the same as the

calibration parameter used to create the mosaic in "Square Targeting" node are not

consistent.

<title>Solution:</title>

*  Other than fix the goniometer, Targeting on these mosaic can still work if the

error does not occur frequently enough to interfere drift management.

*  Check "Leginon/Grid/Settings/Use ?? to move to target" against "Leginon/Square

Targeting/Mosaic Settings/CalibrationParameters". The latter should be changed if the

mosaic is already acquire and what seen on the Web is better than what is in Leginon.

The mosaic atlas can be recreated by clicking the "Create" button.

<title>More Explanation:</title>

*  The web tools and Leginon main program use different mechanism to paste together

the atlas.

The targets Leginon "Grid Targeting" node send out is an even spaced raster.

"Grid" node uses that information and the move type set for the node to calculate the

goniometer position it needs to aim for in order to get to where it really wants to go

to and acquires the image. It is done this way so that it can handle any complex move

type. For example, lets say that the model is p=x^2 so that to get to position p' from

0, the goniometer x needs to be sqrt(p'). "Grid Targeting" send the target as p',

"Grid" figure out that x'=sqrt(p') and send the x value to the scope to move.

When it needs to display the atlas in "Square targeting", Leginon uses the actual

goniometer position of each tile image back calculates the targeted position according

to the model. This has the advantage that if there is a problem with the goniometer

and the stage does not move to where it needs to (such as after a pole touch error),

the tile image will appear at its actual position. In the example above, the image

will appear at p' from the image because from the goniometer position x'=sqrt(p'),

p=x^2=(sqrt(p'))^2.

The webtool, however, pastes the atlas together using the targets saved from "Grid

Targeting". It does not care what you have set in "Square Targeting" node. Therefore,

it will always look right, unless of course the stage did not move to the target when

it acquired the image.

h2. Commonly Why:

* You did not initiate refresh by clicking the "current position" tool

* More than one atlas is of the same label.

<title>Solutions:</title>

*  No user correction can be made with atlas of the same label. It requires

modification of the database.

*  Acquire a new atlas with a different label

h2. Targeting is consistently off in the same direction and

distance

Commonly Why: Image shifts in "hl", "sq", or "gr"presets are not aligned with the

presets at higher mags. The low mag "sq" preset is especially prone to this problem.

***Important: When the targeted image is off, it normally means the image shift of the

preset where the target comes from is off. That is, if hole images appear off, it is not the

image shift of "hl" preset that needs correcting but that of "sq" preset.

Solution:

*  Pause before next hole or square by clicking Hole/Toolbar>Pause button or

Square/Toolbar>Pause button, respectively. Which one to pause depends on what is

the next target.

*  Center the grid at a location that can be recognized by either:

*  Move to the alignment position:

(It may be necessary to adjust the position at the scope after this since

the goniometer can not move back to a position as accurately as we need

it).

*  Leginon/Navigation/Stage Location> Choose the name for the stored

alignment position ("align" if you have been following the setup check

list). and send it "To Scope"

*  scope> move the specimen to a position on the same square, either on a

recognizable object or burn a good hole through ice as preparation for lower mag

preset image shift correction

*  Leginon/Presets Manager> send "fa" or other high mag preset with (0.0,0.0)

image shift "To Scope"

*  Leginon/Navigation> "Acquire" an image. The recognizable object or the burn

hole should be at the center of the image, if not move the grid at the

microscope.

*  Check, adjust, and save the lower mag preset image shift in hole and sq

*  Leginon/Presets Manager> send the preset "To Scope".

*  Leginon/Navigation> "Acquire" an image in the Camera

Configuration.

*  Leginon/Navigation> "Navigate" by "Image Shift" to center on the

reference object or the burn mark made in the high mag presets.

*  Leginon/Presets Manager> save the new image shift by retrieving them

"From Scope"

*"Navigate" means selecting the navigate tool on the top right of image

display and then left-clicking the location of the new center that is translated

by the given TEM Parameter.

*  Leginon/Drift Manager/Toolbar>"Declare Drift"(declare drift icon) to force

target shift check at the next possible time point. This will correct further

targeting.

*  "Continue" data collection on next hole or square target by clicking

Hole/Toolbar> "Continue" button or Square/> "Continue" button,

respectively.

Still not working: Your high mag "fc", "en", or "ef" has an image shift other than that

of "fa". The latter is commonly used in image shift alignment of presets.

Solution:

*  Leginon/Presets Manager> select individual suspect to check its image shift x

and y, change them to the correct values but not send the preset to Scope.

h2. Targeting is consistently off in opposite direction from

where you pick

Commonly Why: Camera configuration is different in calibration and now.

*  All your targeting would be wrong. "Abort" target processing in the Target

Watcher of "squares" and "holes".

*  If you use the Tietz camera: scope/Leginon/Instrument/Camera> check your

camera configuration. mirror should be ['vertical']. Change it if not, and "Set" the

configuration.

*  If you use the Gatan camera: scope/Digital Micrograph> check your camera

configuration.

*  Leginon/Presets Manager> send "sq" or "grid" "To Scope"

*  Leginon/Navigation> "Acquire" an image with "Use this configuration" Unchecked

in the Camera Configuration.

*  Leginon/Navigation> "Navigate" by "Image Shift", "Beam Shift", or "Stage

Position" to center on a reference object or burn mark made in the high mag presets.

This is to check if it behaves correctly now.

*  Select targets again, or, if atlas is wrong, republish the target list to make

the atlas.

h2. Hole targeting is inconsistently off or goes bad in the

next run after correction

Commonly Why: Not well understood but may be related to microscopy alignment and

settings such as normalization of lenses. up to 1.5 um error has been observed even with a

reasonable calibration. The following solutions only works some times.

*  Leginon/Drift Manager>"Declare Drift" to force correction for possible

drift.

*  Repeat image shift correction alignment <link linkend="toffinsamedir">as in the

case when targeting is consistently off</link>.

*  Ignore the first targetted '"hole" in the "squares" image. The second and on are

more consistent when there is a hysteresis problem.

*  Repeat <link linkend="gonm_m_only">"Mag Only" Modeled Stage Calibration</link> at

the "square" preset.

h2. Hole targeting is off more when the image shift target is

away from the center of the image

Commonly Why: Not well understood but may be related to microscopy image shift causes

different physical movement at different magnifications since Leginon uses the image shift

calibration at the lower mag where the target comes from to determine the required image

shift at the higher mag.

No current solution

h2. Automatic hole finding fails to pick up good ice thickness

after set up

Commonly Why:

* Electron beam intensity has changed

* Blob threshold is not inclusive enough to tolerate variations

* Ice contaimination

Solutions

* Monitor electron beam with a saved location with an empty hole in navigator and

check the recorded intensity periodically and adjust zero ice thickness

accordingly.

* Reduce Blob threshold in Alias nodes of the Hole Finder.

* Make sure the anticontamination cryo box is properly inserted on the scope.

* Remove the film camera from the scope if you don't need film.

h2. No broken grid square for dose measurement and ZLP alignment

(MSI-Tomography)

Solution

* Burn a hole clear of ice and pick it as the reference target in "Hole Targeting"

or "Tomography Targeting"