MSI set-up in more details¶

This section shows some of the more important and complicated steps in the set-up checklist. It assumes that the user knows the nomenclatures. See the full documentation if you want the detailed explanation. It also assumes that general set-up and calibrations exist from previous sessions so that most calibrations can remain unchanged. The steps that are NRAMM specific are marked. MSI now features stage Eucentric height correction at each square.

NOTE: The Tietz imaging software (EMMenu) must be turned OFF before Leginon is started. In contrast to this, the Gatan imaging software (Digital Micrograph) must be ON before Leginon is started.

Starting Up¶

Set up is most stable with a good microscope alignment.

1. Initial alignment at the microscope See Chapter on Microscope setup for details.

• scope> Insert specimen holder
• scope> Put stage to eucenter and reset defocus in both HM mode AND LM mode
• scope> Check gun tilt and shift
• scope> Check rotation center and beam tilt pivot points in the HM mode
• scope> Save the goniomenter position for the square with intact supporting film where the alignment was done and do the same for a square where the supporting film is broken (where bright/dark images can be acquired).

2. Start python-based leginon main program amd leginon
client. See Chapter on Start Leginon.

• scope> Set up CCD camera
• scope> Start Leginon Client
• scope> If the following error message appears, DO NOT touch it, or Leginon Client will need restarting

"Exception EAccessViolation in module adaExp.exe at....
Access violation at address xxxx in module 'adaExp.exe'. Read of address xxxx"

• main> start-leginon.py
• Leginon Setup Wizard> Create new or Select old session, connect to the instrument host, and start.

3. Start your preferred "MSI" application.

a.  Leginon/Application/Run> Select the "MSI" application you would like to use (Edge,T,Raster, or Tomography), the scope, and the main launcher. Click Run.
 b.  Leginon/Node Selector> Select "Presets Manager" node.
 c. Leginon/Presets Manager/Preset Selector> Import presets from a previous working session 
  * Select the TEM and camera to "Find" sessions closest to the condition of your data collection.
  * Use shift-key to select a series of presets if needed. Hold-down to ctrl-key allows individual selection/deselection.
  * Click "Import" button to import, the presets should appear in the Presets Manager/Preset Selector window.
  * Click "OK" to exit "Import Presets" window.

Note: Creating new preset and adjusting the preset are covered in Calibration Chapter under [[Creating Presets for the first time|Creating presets the first time]].

d.  The cycle order should match the sequence of data acquisition to minimize hysteresis. If the presets are imported from previous session, the cycle order is imported automatically. As an example, the cycle for naming scheme used here at NRAMM is gr -> sq -> hl -> fa -> fc -> en -> ef.

e. Leginon/Presets Manager> Select a preset in the list box and then click "To Scope"  to apply the settings to the scope and synchronize Leginon and the scope settings.

4. Save the alignment and empty position in Leginon

a.  scope> Recall the stored intact or empty position or go to such
location.

b.  Leginon/Navigation/Toolbar/Stage Locations>  Enter a New name to store Stage Location such as, for example, "empty" and "align", respectively. Save the xy coordinate only. To recall the position, select the named location under the same tool and click on "send to scope".

Grid preset image shift refinement¶

1. Leginon/Presets Manager> Send a preset at an intermediate mag at HM mode as
the reference (e.g. hl) "To Scope"<inlinegraphic
fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
format="PNG"/>

(Note: sq preset in LM mode can be used if the image alignment between LM and HM
is known to be good).

• scope> Move the grid to a position on the "align" square, either on a
  recognizable object or burn a good hole through ice at the center of the CCD as
  preparation for lower mag preset image shift correction.

• Leginon/Presets Manager> Click on the tool "Align presets to each other"
  <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png"
  format="PNG"/> .

• Check, adjust, and save the preset(s) to be aligned.

The left panel displays and navigate by "Stage Position" of the reference preset
while the right panel displays and navigate by "Image Shift" of the preset to be
aligned

• Leginon/Presets Manager/Align Presets> Choose the reference (hl) preset
  on the left panel.

• Leginon/Presets Manager/Align Presets> Choose the aligning (gr) preset
  on the right panel.

• Leginon/Presets Manager/Align Presets> Select "Similar look across mags"
  mode at the top left corner of the window. This will use a smaller part of the
  CCD to acquire the image for the lower mag preset so that it appears to be
  magnified in the 512x512 image panel.

• Leginon/Presets Manager/Align Presets> "Start" image acquisition.

• Leginon/Presets Manager/Align Presets> If the recongnizable object can
  not be found in both panels, go back to step one to choose a lower mag preset or
  a better object.

• Leginon/Presets Manager/Align Presets> If the recongnizable object can
  be found in both panels but not at the center on the reference left panel,
  navigate* it to the center using its "Click Tool". Wait for the move to complete
  and both images refreshed.

• Leginon/Presets Manager/Align Presets> Navigate* the object on the right
  panel to the center using its "Click Tool".The new image shift is automatically
  saved at each move.

• Repeat 7 if necessary.

• "Navigate" means selecting the navigate tool<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/arrow.png" format="PNG"/> on
  the top right of image display and then double clicking the location of the new center that
  is translated by the given TEM Parameter.

Grid preset stage postion refinement¶

Stage position model Scale and Rotation is stable as long as the LM eucentric height
defocus is set to zero before each session, although variation from day to day is normal.
Check the stage movement calibration of gr preset by navigating it in Navigation node.
Within 10% error is satisfactory since reaching a square does not require high accuracy. If
not, perform stage model position scale and rotation
calibration> at gr preset using "Calibration" application as
following:

Why goniometer model is
better.

• scope> Remove the objective apperture.

• Leginon> Run "Calibrations" Application

• Leginon/Presets Manager> Send "gr" preset to scope.

• Leginon/GonioModeling/Settings/Measurement> Set axis=x,
  Points=5,Tolerance=25%, and Interval=1e-05 m. Default model label is the session
  name.

• Leginon/GonioModeling/Settings/Modeling> Set "Scale and Rotation Adjustment
  Only"=Yes. Other settings will be set automatically once measurements are
  finished.

• Leginon/GonioModeling> Click on <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/cam_ruler.png"
  format="PNG"/> will start "Measure" the shifts of between points that are moved on x
  axis of the goniometer.

• Leginon/GonioModeling> Click on <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG"
  /> will "Calibrate" the scale and rotation of the model on x axis of the
  goniometer.

• Leginon/GonioModeling> repeat 4-7 with the axis set to y.

Acquire mosaic atlas of the grid¶

• scope> Remove the objective apperture.

• Leginon/Grid Targeting> Click "Settings" to check the preset used, the size
  and name of the atlas.

• Leginon/Grid Targeting> Click "Calculate Atlas" <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/calculate.png"
  format="PNG"/>

• Leginon/Grid Targeting> Click "Submit Targets" <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG"
  />to begin acquiring the atlas.

• Leginon/Square Targeting> Follow the atlas progress in the display. (***There
  is no sign at the end currently other than that nothing happens for a long time and
  that the atlas dimension is the same in other direction when it is completed.)

• scope> Insert the objective aperture.

• Leginon/Presets Manager/Send an HM preset "To Scope" several times to cycle all
  magnifications and warm up the objective lens to minimize the image shift hysteresis
  in switching between LM and HM modes. If the scope has been in LM mode for a long
  time, wait for at least 30 min before final image shift refinement of needed LM preset
  during acquisition (i.e. sq)

Determine the proper exposure condition for exposure
presets¶

Exposure presets are the base presets for data collection. To keep it to low dose
condition such as 10 electron/Angstrum, measurement of dose is performed and the exposure
time is adjusted. If scope or camera parameters are changed during the session, the dose
value is no longer valid and should be remeasured.

• Leginon/Navigation/Toolbar/Stage Locations<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
  format="PNG"/>> Send the "empty" position "To scope".

• Leginon/Presets Manager/Preset Selector> Select a high mag exposure (i.e. "en"
  or "ef"), confirm that the parameters are what you desire for the experiment, and then
  send "To Scope"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
  format="PNG"/>.

• scope> Expand the beam to at least cover the CCD (> medium circle mark on
  the viewing screen) and to the size you desire.

• Leginon/Presets Manager/Preset Selector> If you have changed the size or
  location of the beam or other preset scope parameters at the scope, you must retrieve
  the preset parameter "From scope"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png"
  format="PNG"/> first before checking and saving the dose value.

• Leginon/Presets Manager/Preset Selector> "Acquire dose image"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/acquire.png"
  format="PNG"/> for the selected preset.

• Leginon/Presets Manager/Dose Image> Check the "Dose" value.

• Leginon/Presets Manager/Dose Image> If desired dose is known, fill in the
  value and then click "Match". This will change exposure time to give a close match of
  the value entered.

• Leginon/Presets Manager/Dose Image> If not satisfied with the value, click
  "No", change the exposure time by editing the preset, and try again (No need to send
  the preset "To scope" again for the repeat)

• If the scope can not give such illumination to satisfy dose, exposure time and
  area requirement, alignment is required or change of gun lens or spot size is
  needed.

• Leginon/Presets Manager> Copy the preset parameter to the other exposure
  preset.

• Preset Selector> send the preset that has the dose measurement "To
  Scope"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
  format="PNG"/>

• Preset Selector> select the preset to recieve the copy

• Preset Selector> retrieve the parameters "From Scope"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png"
  format="PNG"/> for the selected preset.

• Preset Selector/Edit><inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/edit.png"
  format="PNG"/> Modify defocus for the preset after the retrieval.

Update bright/dark correction images for exposure
presets¶

Exposure presets are the base presets for data collection. To make sure the best
correction is applied to them, the correction images should be acquired prior to the
experiment at session. Since no correlation is normally done on these images, only one channel is needed. Normalization image might need to be
Bright and Dark reference images if a large chunk of region is blocked by a
fallen debris.

• Leginon/Presets Manager> Select a dose measured high mag exposure (i.e. "en"
  or "ef"), and then send "To Scope".

• Leginon/Correction/Settings>

• Select TEM and camera to match that will be used.

• Camera Configuration: The exposure preset camera configuration

• Leginon/Correction/Settings> Click OK to exit settings.

• Leginon/Correction/Toolbar> Select "Raw image " from the pull down list of
  acquisition modes and then click on "Acquire" button next to the selector to view an
  image that is not corrected.

• Leginon/Correction/Toolbar> Select "Dark reference" in the acquisition mode
  selector and then click "Acquire" to acquire the Dark reference image for this
  particular camera configuration.

• Leginon/Correction/Toolbar> Select "Bright reference" and repeat the
  acquisition to obtain the Bright reference.

• Leginon/Correction/Toolbar> Select "Corrected image" and then "Acquire" to
  view the corrected image. A corrected image should be free of artifacts and have
  smaller standard deviation than the raw image, in general.

• Leginon/Correction/Toolbar> If there are regions consistently over normalizes
  the acquired image, either select the region and use the "+" tool to "Add Region to
  Bad Pixel List", or click on<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
  format="PNG"/>to "Add Extreme Points to Bad Pixel List" one of more times to find
  the outliers. Right click on a shown bad pixel removes it from the list.

Medium mag preset image and beam shift refinement¶

Preset image shift alignment can be done follow the same procedure as the section "Grid preset image shift refinement" but is more
efficient using the automated mode as follows.

• Leginon/Presets Manager> Send one of the exposure preset as the reference
  (e.g. en) "To Scope"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
  format="PNG"/>

• scope> Move the grid to a position on the "align" square, either on a
  recognizable object or burn a good hole through ice at the center of the CCD as
  preparation for lower mag preset image shift correction.

• Leginon/Presets Manager> Click on the tool "Align presets to each other"
  <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png"
  format="PNG"/> . The tool automatically select the reference preset that was
  selected in the Presets Manager mainpanel and choose a preset at closest magnification
  as the starting aligning preset.

• Leginon/Presets Manager/Align Presets> Click "Start" to acquire the first
  image pair.

• Check, adjust, and save the preset(s) to be aligned.

The left panel displays and navigate by "Stage Position" of the reference preset
while the right panel displays and navigate by "Image Shift" of the preset to be
aligned

• Leginon/Presets Manager/Align Presets> Changing Selection of "Similar
  look across mags" or "Full CCD" mode at the top left corner of the window will
  cause reacquisition of the image pair in the automated mode.

• Leginon/Presets Manager/Align Presets> If the recongnizable object can
  be found in both panels but not at the center on the reference left panel,
  navigate* it to the center using its "Click Tool". Wait for the move to complete
  and both images refreshed.

• Leginon/Presets Manager/Align Presets> Navigate* the object on the right
  panel to the center using its "Click Tool". The new image shift is automatically
  saved after each move.

• Repeat 3 if necessary.

• Leginon/Presets Manager/Align Presets> Click "Continue" to save the
  alignment to all presets of the same magnification and continue to the next
  preset pair.

• Repeat the process if so desired.

Go to MSI Operation section¶

The setup that requires you to be next to the microscope is completed. Go to the section
on MSI Operation You can come back and continue on this section after you
have learned how to go through the operation without automated hole finding.

Hole Targeting Set-up¶

We can use any image that is already loaded into Hole Targeting to adjust these
parameters. The values in this node vary so much from grid to grid that the best way to
find these parameters is by going through a step-by-step trial-and-error process. To see the
final acquisition and focus targets, enable only the Original, acquisition, and focus
display selections. Refer to Introduction Chapter on [[MSI set-up in more details

|how the various tools behave in the control panel]].

The loaded image normally comes from the image acquired through "Square" node when you
submit targets on the grid atlas in "Square Targeting" node. You can also do a practice run
using the default image comes with Leginon. Leave the filename entry blank when you click
"Load" in the hole finding settings window for the step "Original". The default values in
the node gives an example of reasonable target finding with such default image.

Setting up Hole Targeting Depends on which MSI flavor you are running:

• MSI-Edge uses template matching of the
  edge image

• MSI-T uses template matching of the original
  image

• MSI-Raster creates raster points from a given
  origin

• MSI-Tomography does not use automatic
  target finding. Therefore, it does not requires set up.

Exposure Targeting Set-up¶

Like hole targeting, we can use any image that is already loaded into Exposure Targeting
to adjust these parameters.

Setting up Exposure Targeting Depends on which MSI flavor you are running.

• MSI-Edge uses template matching of the
  edge image

• MSI-T uses template matching of the original
  image

• MSI-Raster creates raster points from a given
  origin

• MSI-Tomography does not use automatic
  target finding. Therefore, it does not requires set up.

Autofocus Test¶

There are two focusers in MSI. "Z Focus" node usually moves stage in Z direction to
Eucentric height at lower mags. "Focus" node usually just correct the defocus in the way
equivalent to turning the focus adjustment knob on the scope at a higher mag. The defocus
measurments in both nodes are based on the same principle of beam-tilt induced image shift.
A correlation (normally phase) is used to obtain the image shift. Therefore, it is important
to test and preform autofocus measurement at area with recognizable feature that does not
move significantly during the defocus measurement.

The test use "Manual_after" focus step in the focus sequence for checking. Two
procedures can be used to reach a area for good correlation measurements:

• Using Simulate Target tool

• Leginon/Presets Manager> Send the autofocus preset "To Scope"

• scope> Move to an area that has recognizable feature on the carbon
  support at the center.

• Leginon/(Z) Focus/Focus Sequence<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png"
  format="PNG"/>> Enable desired autofocus and "Manual_after" focus
  steps.

• Leginon/(Z) Focus/Toolbar> Click Simulate Target<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/simulatetarget.png"
  format="PNG"/> and follow the autofocus

• Submiting focus target from Hole Targeting

*To use this procedure, there has to be a grid mosaic, and the Hole Targeting
should allow user verification of targets

• Leginon/(Z) Focus/Focus Sequence<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png"
  format="PNG"/>> Enable desired autofocus and "Manual_after" focus
  steps.

• Leginon/Square Targeting> Select a test square and submit it<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png"
  format="PNG"/>

• Leginon/Hole Targeting> Submit a focus target to test autofocus in Z Focus
  node and an acquisition target for the next stage. Both targets should be in an
  area that has recognizable feature with carbon support.

• Leginon/Z focus> follow the autofocus test in this node.

• Leginon/Exposure Targeting> Submit a focus target at a location that has
  recognizable feature with carbon support to test autofocus in Focus node.

• Leginon/Focus> follow autofocus test in this node.

Checking the performance of autofocus by the following
criteria:

• During autofocusing

• Display the image and correlection peak: The observed movement of images
  upon beam tilt should be consistent with the correlation peak and the edge of
  the beam should not be visible in any of the beam-tilt images.

• Display the correlation image: The correlation peak at its correct position
  should be significantly higher than background.

• During manual focusing

Manual Focusing can be activated either by enabling "Manual_before/Manual_after"
steps or directly when leginon is idle using (Z) Focus/Toolbar/MF. To activate manual
focus directly, it is necessary to send to scope the preset of the choice (normally
fc) first, though.

• MF> The FFT should show visible and isotropic Thon ring according to the
  fc preset defocus.

• MF/Toolbar> Make sure the move type is "Defocus" and the set value in the
  box is 0 m then click "Set instrument" botton<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
  format="PNG"/>. This will set the defocus to the current 0.

• MF> The perfect autofocus result is that the FFT indicates that the scope
  is at Gaussian focus.

• For relative movement, press "+" or "-". The step size is determined in
  MF/Toolbar/Settings>

• To reset defocus to zero at the new focus, press the so-named
  button<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentsetnew.png"
  format="PNG"/>.

• Clicking the Stop button<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/stop.png"
  format="PNG"/> exits the maunal check.

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