Leginon

h1. MSI Operation

h2.  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.

h2.  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"

h2.  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.

h2.  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.

h2.  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"

h2.  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.

h2.  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.

h2.  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>.