MSI Operation¶

Start Leginon & MSI (if not already started)¶

• local> start-leginon.py

• Leginon Session> log in and select "Create session" if a new session is
  needed.

Create Session> Fill in needed information such as session name and comments.
Select instrument and project, if project database is used. Image path is
automatically selected.

• Leginon Session> Select the session you have just created or use a previous
  session to start.

• Leginon/Application/Run> Select the "MSI" application, the scope, and the
  local launcher. Click Run.

Reload the Atlas (Only if Leginon is being restarted)¶

<important>
The Atlas should have already been created by following the <link
linkend="MISQuickStart">MSI everyday set-up checklist</link>.

</important>

Reload the atlas image of the grid. This is used if Leginon was stopped and
restarted.

• Leginon/Square Targeting/Mosaic <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/atlasmaker.png"
  format="PNG"/>> Increase the size of the mosaic atlas loaded into the image
  display for better resolution. For example, Scale image to: 2048

• Leginon/Square Targeting/Tiles<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/tiles.png"
  format="PNG"/>> Select the atlas label in "Load tiles from mosaic."

• Leginon/Square Targeting/Tiles> Click "Load"

Select Squares on the atlas¶

Pick squares to collect data from by making selections on the atlas.

• Leginon/Square Targeting> Refresh the target display by clicking
  <inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/refresh.png"
  format="PNG"/>.

• Leginon/Square Targeting> Click the "acquisition" selection
  button<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/arrow.png"
  format="PNG"/>.

• Navigate around the atlas to find suitable squares for acquisition.

This step is purely empirical at this point (by eye). If using Quantifoil grids
with ice, look for squares that have holes paler than the surrounding carbon. Although
Leginon has automatic algorithm for square finding, the adjustment of parameters takes
more time than what human can accomplish easily. Use the zoom to look at the holes
more closely. Note that the zoom will take a long time on such a large image.
Therefore...

NOTE: Be sure to be patient when clicking the zoom button, otherwise Leginon will
take a while to catch up to the number of zooms that have been asked for.

• Click on squares that will be acquired.

• red cross hair = previously targeted or "done" squares.

• green cross hair = new targets

• orange cross hair = current postion (updated by pressing the Leginon/Square
  Targeting> Refresh button<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/refresh.png"
  format="PNG"/>)

• Once all the targeted squares have been selected, click "Submit Targets."

• Leginon will indicate where the next step is by showing active green icons next
  to the nodes that are being used. A question mark icon will appear when Leginon
  requires user interation. The message log for each node indicates what is happening
  that particular node.

Select Holes on the first image of a square¶

Pick holes to collect data from by making selections on the first image of a square.

• Leginon/Hole Targeting> Check whether holes were selected (<anchor
  id="MSI_target_color"/>acquisition targets = green crosshair, focus target = blue
  crosshair).

• Edit the targets by mouse left click (add) or right
  click (delete) on the target in the image viewer with the picking tool for the target
  type active.

• If the hole selection is acceptable, uncheck "Allow for user verification of
  picked holes" in Leginon/Hole Targeting/Settings>.

• If the hole selection is not acceptable, try adjusting the Hole Targeting node to
  find holes in the current image of a square. Refer to the <link
  linkend="holetargeting_setup">Hole Targeting Set-up</link>.

• Click "Submit Targets"

h2.
Manually check focuser that uses Stage Z as the correction
method

Manually checking the focus after eucentric focusing of hl images is recommended for
bent grids.

• When the Manual Focus window opens automatically (when Leginon reaches the Z
  Focus node), click "Eucentric focus to instrument"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
  format="PNG"/>. The rings of the CTF in the FFT should disappear if it is looking at
  the carbon support using fc preset. If hl preset is used to look at a hole during
  manual focus, look for the loss of contrast in IMAGE.

• Adjust "Stage Z" until the image appears to be focused if not already so. Use a
  large step such as 5e-6 m.

• If the defocus is consistently off by more than +/- 5 um., then the <link
  linkend="btcal_def">Beam Tilt calibrations</link> (defocus) may need to be
  repeated.

Make sure that the stage is at eucentric height in the calibration and that the
<link linkend="microscope_setup">microscope has been well aligned</link>.

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

• Leginon/Z Focus/Focus Sequence> Disable "Manual_after" focus step when it is
  not required.

Select Exposure and Focus targets on the first image of a hole¶

Pick exposure and focus targets on the first image of a hole.

• Leginon/Exposure Targeting> Click the Original, acquisition, and focus
  selections in the image control panel.

• Check whether exposure (green crosshair) and focus (blue crosshair) targets were
  selected correctly.

• Edit the targets by mouse left click (add) or right click (delete) on the target
  in the image viewer with the picking tool for the target type active.

• If the exposure and focus selection is acceptable, uncheck "Allow for user
  verification of picked holes" in Leginon/Exposure Targeting/Settings>.

• If the exposure and focus selection is not acceptable, try adjusting the Hole
  Finder to pick the correct targets. Refer to <link linkend="exptargeting_setup"

Exposure Targeting Set-up</link>

• Click "Submit Targets"

Manually check focuser that uses Defocus as the correction method¶

Manually check the focus after the first high magnification images that are acquired.
Then turn off "manual_after" focus step once it has been verified that the autofocus routine
is properly correcting the defocus and astigmatism.

• When the Manual Focus window opens automatically (when Leginon reaches the Focus
  node), click "Set Instrument"<inlinegraphic
  fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
  format="PNG"/> to send what Leginon thinks is zero defocus to the microscope. The
  rings of the CTF in the FFT should disappear once the defocus is at zero.

• If the rings are still visable or are astigmatic in the FFT, then the <link
  linkend="btcal_def">Beam Tilt calibrations</link> (defocus and stimator) may need to
  be repeated.

Make sure that the stage is at eucentric height and that the <link
linkend="microscope_setup">microscope has been well aligned</link>.

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

• Leginon/Focus/Focus Sequence> Disable "Manual_after" focus step when it is not
  required.

View the images online¶

The images Leginon has acquired and published to the database can be viewed through the
online viewers, the Leginon Image Viewer, the Leginon 3-Way Viewer, and the Leginon Observer
Interface (LOI) which displays the currently acquired images.

• Go to http://yourhost/dbem and open either the Leginon Image Viewer, the Leginon
  3-Way Viewer, or the LOI.

• Select today's Session from the pull-down list.

• Have fun browsing through your images! You can view the parent images of images
  selected in the main viewer of the 3-way viewer in the smaller views by specify the
  appropriate preset.

Interruption¶

Targets that are submitted can no longer be modified individually from the node where
you selected them. If you need to abort the unprocessed targets in a list, you need to go to
the node that recieve it and click on the "Abort" button. In other words, if you want to
abort all remaining holes in a square, you need to abort at the "Hole" node.

Same is true with pausing. Pausing is usually used when the cryo stage requires refill.
To allow proper tracking of targets and drift, we recommand that you pause at "Hole" node
for the purpose if exposure targets are not queued.

More information about interrupting data collection can be found in the <link
linkend="MSI_pause">Trouble Shooting Chapter regarding pausing and aborting during data
collection</link>.