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MSI set up in more details » History » Version 4

Alex Kadokura, 06/04/2010 03:53 PM

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h1. MSI set-up in more details
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h2. Starting Up
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Set up is most stable with a good microscope alignment.
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*  Initial alignment at the microscope See Chapter on [[Microscope Set-up|Microscope setup]] for details.
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*  scope> Insert specimen holder
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*  scope> Put stage to eucenter and reset defocus in both HM mode AND LM
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mode
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*  scope> Check gun tilt and shift
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*  scope> Check rotation center and beam tilt pivot points in the HM
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mode
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*  scope> Save the goniomenter position for the square with intact
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supporting film where the alignment was done and do the same for a square where
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the supporting film is broken (where bright/dark images can be acquired).
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* Start python-based leginon main program amd leginon
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client. See Chapter on [[Microscope Set-up|Start Leginon]].
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*  scope> Set up [[MSI set-up in more details#Starting-Up|CCD camera]]
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* [[Run Leginon Client on the Microscope Computer|scope> Start Leginon Client]]
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*  scope> If the error message
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<blockquote>
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_"Exception EAccessViolation in module adaExp.exe at
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...._
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_Access violation at address xxxx in module 'adaExp.exe'. Read of
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address xxxx"_
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</blockquote>
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appears, DO NOT touch it, or Leginon Client will need restarting
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* [[Start Leginon at your main working station remotely|main> start-leginon.py]]
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*  Leginon Setup Wizard> Create new or Select old session, connect to the
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instrument host, and start.
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*  Start your preferred "MSI" application.
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*  Leginon/Application/Run> Select the "MSI" application you would like to
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use (Edge,T,Raster, or Tomography), the scope, and the main launcher. Click
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Run.
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*  Leginon/Node Selector> Select "Presets Manager" node.
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* Leginon/Presets Manager/Preset Selector> Import
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presets from a previous working session <inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/import.png"
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format="PNG"/>
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* Select the TEM and camera to "Find" sessions closest to the condition of
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your data collection.
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* Use shift-key to select a series of presets if needed. Hold-down to
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ctrl-key allows individual selection/deselection.
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* Click "Import" button to import, the presets should appear in the
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Presets Manager/Preset Selector window.
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* Click "OK" to exit "Import Presets" window.
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* Creating new preset and adjusting the preset are covered in Calibration
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Chapter under [[Creating Presets for the first time|Creating presets the first
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time]].
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*  The cycle order should match the sequence of data acquisition to minimize
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hysterisis. If the presets are imported from previous session, the cycle order
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is imported automatically. As an example, the cycle for naming scheme used here
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at NRAMM is gr -> sq -> hl -> fa -> fc -> en -> ef.
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* Leginon/Presets Manager> Select a preset in
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the list box and then click "To Scope" <inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
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format="PNG"/> to apply the settings to the scope and synchronize Leginon and
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the scope settings.
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* Save the alignment and empty position in Leginon
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*  scope> Recall the stored intact or empty position or go to such
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location.
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*  Leginon/Navigation/Toolbar/Stage Locations>
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<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
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format="PNG"/> Enter a New name to store Stage Location such as, for example,
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"empty" and "align", respectively. Save the xy coordinate only. To recall the
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position, select the named location under the same tool and click on "send to
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scope".
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h2. Grid preset image shift refinement
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*  Leginon/Presets Manager> Send a preset at an intermediate mag at HM mode as
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the reference (e.g. hl) "To Scope"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
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format="PNG"/>
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(Note: sq preset in LM mode can be used if the image alignment between LM and HM
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is known to be good).
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*  scope> Move the grid to a position on the "align" square, either on a
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recognizable object or burn a good hole through ice at the center of the CCD as
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preparation for lower mag preset image shift correction.
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*  Leginon/Presets Manager> Click on the tool "Align presets to each other"
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<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png"
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format="PNG"/> .
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*  Check, adjust, and save the preset(s) to be aligned.
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The left panel displays and navigate by "Stage Position" of the reference preset
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while the right panel displays and navigate by "Image Shift" of the preset to be
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aligned
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*  Leginon/Presets Manager/Align Presets> Choose the reference (hl) preset
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on the left panel.
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*  Leginon/Presets Manager/Align Presets> Choose the aligning (gr) preset
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on the right panel.
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*  Leginon/Presets Manager/Align Presets> Select "Similar look across mags"
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mode at the top left corner of the window. This will use a smaller part of the
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CCD to acquire the image for the lower mag preset so that it appears to be
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magnified in the 512x512 image panel.
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*  Leginon/Presets Manager/Align Presets> "Start" image acquisition.
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*  Leginon/Presets Manager/Align Presets> If the recongnizable object can
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not be found in both panels, go back to step one to choose a lower mag preset or
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a better object.
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*  Leginon/Presets Manager/Align Presets> If the recongnizable object can
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be found in both panels but not at the center on the reference left panel,
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navigate* it to the center using its "Click Tool". Wait for the move to complete
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and both images refreshed.
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*  Leginon/Presets Manager/Align Presets> Navigate* the object on the right
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panel to the center using its "Click Tool".The new image shift is automatically
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saved at each move.
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*  Repeat 7 if necessary.
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* "Navigate" means selecting the navigate tool<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/arrow.png" format="PNG"/> on
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the top right of image display and then double clicking the location of the new center that
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is translated by the given TEM Parameter.
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h2. Grid preset stage postion refinement
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Stage position model Scale and Rotation is stable as long as the LM eucentric height
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defocus is set to zero before each session, although variation from day to day is normal.
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Check the stage movement calibration of gr preset by navigating it in Navigation node.
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Within 10% error is satisfactory since reaching a square does not require high accuracy. If
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not, perform stage model position _[[Modeled Stage Position calibration#Magnification-and-image-rotation-adjustment-of-the-model|scale and rotation
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calibration]]> at gr preset_ using "Calibration" application as
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following:
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_[[Modeled Stage Position calibration|Why goniometer model is
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better]]._
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*  scope> Remove the objective apperture.
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*  Leginon> Run "Calibrations" Application
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*  Leginon/Presets Manager> Send "gr" preset to scope.
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*  Leginon/GonioModeling/Settings/Measurement> Set axis=x,
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Points=5,Tolerance=25%, and Interval=1e-05 m. Default model label is the session
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name.
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*  Leginon/GonioModeling/Settings/Modeling> Set "Scale and Rotation Adjustment
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Only"=Yes. Other settings will be set automatically once measurements are
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finished.
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*  Leginon/GonioModeling> Click on <inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/cam_ruler.png"
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format="PNG"/> will start "Measure" the shifts of between points that are moved on x
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axis of the goniometer.
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*  Leginon/GonioModeling> Click on <inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG"
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/> will "Calibrate" the scale and rotation of the model on x axis of the
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goniometer.
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*  Leginon/GonioModeling> repeat 4-7 with the axis set to y.
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h2. Acquire mosaic atlas of the grid
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*  scope> Remove the objective apperture.
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*  Leginon/Grid Targeting> Click "Settings" to check the preset used, the size
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and name of the atlas.
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* Leginon/Grid Targeting> Click "Calculate Atlas" <inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/calculate.png"
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format="PNG"/>
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*  Leginon/Grid Targeting> Click "Submit Targets" <inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png" format="PNG"
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/>to begin acquiring the atlas.
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*  Leginon/Square Targeting> Follow the atlas progress in the display. (***There
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is no sign at the end currently other than that nothing happens for a long time and
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that the atlas dimension is the same in other direction when it is completed.)
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*  scope> Insert the objective aperture.
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*  Leginon/Presets Manager/Send an HM preset "To Scope" several times to cycle all
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magnifications and warm up the objective lens to minimize the image shift hysteresis
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in switching between LM and HM modes. If the scope has been in LM mode for a long
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time, wait for at least 30 min before final image shift refinement of needed LM preset
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during acquisition (i.e. sq)
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h2. Determine the proper exposure condition for exposure
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presets
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Exposure presets are the base presets for data collection. To keep it to low dose
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condition such as 10 electron/Angstrum, measurement of dose is performed and the exposure
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time is adjusted. If scope or camera parameters are changed during the session, the dose
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value is no longer valid and should be remeasured.
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*  Leginon/Navigation/Toolbar/Stage Locations<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
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format="PNG"/>> Send the "empty" position "To scope".
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*  Leginon/Presets Manager/Preset Selector> Select a high mag exposure (i.e. "en"
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or "ef"), confirm that the parameters are what you desire for the experiment, and then
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send "To Scope"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
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format="PNG"/>.
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*  scope> Expand the beam to at least cover the CCD (> medium circle mark on
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the viewing screen) and to the size you desire.
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*  Leginon/Presets Manager/Preset Selector> If you have changed the size or
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location of the beam or other preset scope parameters at the scope, you must retrieve
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the preset parameter "From scope"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png"
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format="PNG"/> first before checking and saving the dose value.
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*  Leginon/Presets Manager/Preset Selector> "Acquire dose image"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/acquire.png"
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format="PNG"/> for the selected preset.
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*  Leginon/Presets Manager/Dose Image> Check the "Dose" value.
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*  Leginon/Presets Manager/Dose Image> If desired dose is known, fill in the
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value and then click "Match". This will change exposure time to give a close match of
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the value entered.
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*  Leginon/Presets Manager/Dose Image> If not satisfied with the value, click
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"No", change the exposure time by editing the preset, and try again (No need to send
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the preset "To scope" again for the repeat)
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*  If the scope can not give such illumination to satisfy dose, exposure time and
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area requirement, alignment is required or change of gun lens or spot size is
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needed.
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*  Leginon/Presets Manager> Copy the preset parameter to the other exposure
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preset.
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* Preset Selector> send the preset that has the dose measurement "To
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Scope"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
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format="PNG"/>
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* Preset Selector> select the preset to recieve the copy
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* Preset Selector> retrieve the parameters "From Scope"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentget.png"
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format="PNG"/> for the selected preset.
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* Preset Selector/Edit><inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/edit.png"
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format="PNG"/> Modify defocus for the preset after the retrieval.
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h2. Update bright/dark correction images for exposure
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presets
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Exposure presets are the base presets for data collection. To make sure the best
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correction is applied to them, the correction images should be acquired prior to the
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experiment at session. Since no correlation is normally done on these images, only one [[Bright and Dark reference images#Correction-Channels|channel]] is needed. Normalization image might need to be
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[[Bright and Dark reference images#Correction-Planmodified]] if a large chunk of region is blocked by a
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fallen debris.
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*  Leginon/Presets Manager> Select a dose measured high mag exposure (i.e. "en"
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or "ef"), and then send "To Scope".
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*  Leginon/Correction/Settings>
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*  Select TEM and camera to match that will be used.
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*  Camera Configuration: The exposure preset camera configuration
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*  Leginon/Correction/Settings> Click OK to exit settings.
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*  Leginon/Correction/Toolbar> Select "Raw image " from the pull down list of
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acquisition modes and then click on "Acquire" button next to the selector to view an
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image that is not corrected.
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*  Leginon/Correction/Toolbar> Select "Dark reference" in the acquisition mode
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selector and then click "Acquire" to acquire the Dark reference image for this
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particular camera configuration.
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*  Leginon/Correction/Toolbar> Select "Bright reference" and repeat the
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acquisition to obtain the Bright reference.
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*  Leginon/Correction/Toolbar> Select "Corrected image" and then "Acquire" to
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view the corrected image. A corrected image should be free of artifacts and have
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smaller standard deviation than the raw image, in general.
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*  Leginon/Correction/Toolbar> If there are regions consistently over normalizes
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the acquired image, either select the region and use the "+" tool to "Add Region to
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Bad Pixel List", or click on<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/stagelocations.png"
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format="PNG"/>to "Add Extreme Points to Bad Pixel List" one of more times to find
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the outliers. Right click on a shown bad pixel removes it from the list.
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h2. Medium mag preset image and beam shift
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refinement
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Preset image shift alignment can be done follow the same procedure as the section "[[MSI set-up in more details#Grid-preset-image-shift-refinement|Grid preset image shift refinement]]" but is more
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efficient using the automated mode as follows.
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*  Leginon/Presets Manager> Send one of the exposure preset as the reference
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(e.g. en) "To Scope"<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
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format="PNG"/>
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*  scope> Move the grid to a position on the "align" square, either on a
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recognizable object or burn a good hole through ice at the center of the CCD as
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preparation for lower mag preset image shift correction.
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*  Leginon/Presets Manager> Click on the tool "Align presets to each other"
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<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/alignpresets.png"
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format="PNG"/> . The tool automatically select the reference preset that was
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selected in the Presets Manager mainpanel and choose a preset at closest magnification
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as the starting aligning preset.
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*  Leginon/Presets Manager/Align Presets> Click "Start" to acquire the first
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image pair.
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*  Check, adjust, and save the preset(s) to be aligned.
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The left panel displays and navigate by "Stage Position" of the reference preset
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while the right panel displays and navigate by "Image Shift" of the preset to be
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aligned
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*  Leginon/Presets Manager/Align Presets> Changing Selection of "Similar
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look across mags" or "Full CCD" mode at the top left corner of the window will
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cause reacquisition of the image pair in the automated mode.
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*  Leginon/Presets Manager/Align Presets> If the recongnizable object can
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be found in both panels but not at the center on the reference left panel,
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navigate* it to the center using its "Click Tool". Wait for the move to complete
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and both images refreshed.
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*  Leginon/Presets Manager/Align Presets> Navigate* the object on the right
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panel to the center using its "Click Tool". The new image shift is automatically
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saved after each move.
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*  Repeat 3 if necessary.
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*  Leginon/Presets Manager/Align Presets> Click "Continue" to save the
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alignment to all presets of the same magnification and continue to the next
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preset pair.
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*  Repeat the process if so desired.
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h2. Go to MSI Operation section
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The setup that requires you to be next to the microscope is completed. Go to the section
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on [[MSI Operation|MSI Operation]].
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h2. Hole Targeting Set-up
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We can use any image that is already loaded into Hole Targeting to adjust these
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parameters.  The values in this node vary so much from grid to grid that the best way to
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find these parameters is by going through a step-by-step trial-and-error process. To see the
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final acquisition and focus targets, enable only the Original, acquisition, and focus
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display selections. Refer to Introduction Chapter on [[MSI set-up in more details
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|how the various tools behave in the control panel]].
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The loaded image normally comes from the image acquired through "Square" node when you
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submit targets on the grid atlas in "Square Targeting" node.  You can also do a practice run
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using the default image comes with Leginon. Leave the filename entry blank when you click
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"Load" in the hole finding settings window for the step "Original". The default values in
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the node gives an example of reasonable target finding with such default image.
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Setting up Hole Targeting Depends on which MSI flavor you are running:
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* [[Hole Targeting Set-up for MSI-Edge|MSI-Edge]] uses template matching of the
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edge image
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* [[JAHC finder nodes set-up|MSI-T]] uses template matching of the original
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image
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* [[raster finder nodes set-up|MSI-Raster]] creates raster points from a given
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origin
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* [[Initial MSI application preference setup#Settings-that-are-the-same-as-in-other-MSI-applications-in-queuing-mode|MSI-Tomography]] does not use automatic
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target finding. Therefore, it does not requires set up.
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h2.  Exposure Targeting Set-up
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Like hole targeting, we can use any image that is already loaded into Exposure Targeting
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to adjust these parameters.
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Setting up Exposure Targeting Depends on which MSI flavor you are running.
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* [[Hole Targeting Set-up for MSI-Edge|MSI-Edge]] uses template matching of the
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edge image
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* [[JAHC finder nodes set-up|MSI-T]] uses template matching of the original
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image
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* [[raster finder nodes set-up|MSI-Raster]] creates raster points from a given
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origin
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* [[Initial MSI application preference setup#Settings-that-are-the-same-as-in-other-MSI-applications-in-queuing-mode|MSI-Tomography]] does not use automatic
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target finding. Therefore, it does not requires set up.
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h2. Autofocus Test
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There are two focusers in MSI. "Z Focus" node usually moves stage in Z direction to
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Eucentric height at lower mags. "Focus" node usually just correct the defocus in the way
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equivalent to turning the focus adjustment knob on the scope at a higher mag. The defocus
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measurments in both nodes are based on the same principle of beam-tilt induced image shift.
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A correlation (normally phase) is used to obtain the image shift. Therefore, it is important
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to test and preform autofocus measurement at area with recognizable feature that does not
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move significantly during the defocus measurement.
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The test use "Manual_after" focus step in the focus sequence for checking. Two
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procedures can be used to reach a area for good correlation measurements:
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*  Using Simulate Target tool
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*  Leginon/Presets Manager> Send the autofocus preset "To Scope"
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*  scope> Move to an area that has recognizable feature on the carbon
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support at the center.
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*  Leginon/(Z) Focus/Focus Sequence<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png"
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format="PNG"/>> Enable desired autofocus and "Manual_after" focus
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steps.
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*  Leginon/(Z) Focus/Toolbar> Click Simulate Target<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/simulatetarget.png"
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format="PNG"/> and follow the autofocus
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*  Submiting focus target from Hole Targeting
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*To use this procedure, there has to be a grid mosaic, and the Hole Targeting
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should allow user verification of targets
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*  Leginon/(Z) Focus/Focus Sequence<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/focus_sequence.png"
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format="PNG"/>> Enable desired autofocus and "Manual_after" focus
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steps.
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*  Leginon/Square Targeting> Select a test square and submit it<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/play.png"
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format="PNG"/>
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*  Leginon/Hole Targeting> Submit a focus target to test autofocus in Z Focus
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node and an acquisition target for the next stage. Both targets should be in an
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area that has recognizable feature with carbon support.
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*  Leginon/Z focus> follow the autofocus test in this node.
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*  Leginon/Exposure Targeting> Submit a focus target at a location that has
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recognizable feature with carbon support to test autofocus in Focus node.
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*  Leginon/Focus> follow autofocus test in this node.
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Checking the performance of autofocus by the following
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criteria:
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*  During autofocusing
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*  Display the image and correlection peak: The observed movement of images
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upon beam tilt should be consistent with the correlation peak and the edge of
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the beam should not be visible in any of the beam-tilt images.
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*  Display the correlation image: The correlation peak at its correct position
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should be significantly higher than background.
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*  During manual focusing
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Manual Focusing can be activated either by enabling "Manual_before/Manual_after"
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steps or directly when leginon is idle using (Z) Focus/Toolbar/MF. To activate manual
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focus directly, it is necessary to send to scope the preset of the choice (normally
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fc) first, though.
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*  MF> The FFT should show visible and isotropic Thon ring according to the
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fc preset defocus.
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*  MF/Toolbar> Make sure the move type is "Defocus" and the set value in the
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box is 0 m then click "Set instrument" botton<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentset.png"
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format="PNG"/>. This will set the defocus to the current 0.
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*  MF> The perfect autofocus result is that the FFT indicates that the scope
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is at Gaussian focus.
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*  For relative movement, press "+" or "-". The step size is determined in
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MF/Toolbar/Settings>
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*  To reset defocus to zero at the new focus, press the so-named
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button<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/instrumentsetnew.png"
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format="PNG"/>.
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*  Clicking the Stop button<inlinegraphic
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fileref="http://emg.nysbc.org/software/leginon/images/icons/stop.png"
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format="PNG"/> exits the maunal check.
824 2 Amber Herold
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______
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[[MSI Quick-start|< MSI Quick-start]] | [[Special Operation setup|Special Operation setup >]]
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______