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SerialEM Use

Serial EM provided by David Mastronarde and colleagues from Univ. of Colorado, Boulder

Contents

SerialEmNotes -- older notes, somewhat out of date

SerialEMMacros -- Useful macros

Low Dose Mode in SerialEM

In general, do not run SerialEM low-dose mode at the same time as microscope low-dose software (Tecnai Low Dose or JEOL iMDS)
If for some reason, you do want to use SerialEM low dose while microscope low-dose software is running, be sure that you are in Exposure mode
- Do not change to Search or Focus while running SerialEM low-dose, or everything will be completely messed up and the microscope (and SerialEM) will need to be realigned

Setting up Low-dose mode

Enable SerialEM low-dose while microscope screen is up
After enabled, be sure that "Area to show when screen is down" is set to "Record"
Manually lower the screen
Microscope setting will revert to the previously programmed "Record" mode
If settings need to be changed (magnfication, beam size, beam centering), program it as follows
- Enable the Checkbox "Continuous update of mag and beam"
- Set up microscope as you like, with desired mag, beam size
- When happy, uncheck "Continuous update of mag and beam". Otherwise, you risk accidentally reprogramming it.
Go on to other modes, as follows
If you think the other modes are likely to be messed up, you can click buttons to copy current mode to "View", "Focus" and "Trial". This way, you reprogram from scratch
Under "Area to show when Screen down", select "View"
- Microscope settings will likely change, unless you copied Record mode to View in the previous step
Again, check "Continuous update of mag and beam" and set up the microscope
- Suggest lower end of SA mag (5K or less, but note that mags <5K are problematic on J3200 because of lens series change in this range)
- Beam can be fairly dim, since you will probably use a binning of 4
- Probably best to use same spotsize as Record in order to minimize hysteresis
- Do not worry about beam centering, this will be fixed in a later step
- When done, uncheck "Continuous update" . Also a good idea to take a test View image
Under "Area to show when screen down", select "Focus"
- Check "Continuous update..." as before, set up microscope, then uncheck
- Should use a mag close to the Record mag, though if Record is high (say 50K), probably best not to go above 25K.
- Again, do not adjust centering of beam
If "Keep Focus and Trial Identical" is checked, you are done
Otherwise, set up Trial in the same way as described above

Adjusting the Beam Center

Under "Area to show when screen down", do several cycles of View Focus Record, until beam movement settles down
- suggest to have "Normalize beam through Focus" checked
After a few cycles, go to Record mode and center the beam. This is the "reference" center
Go to View mode. If beam is off center, do the following:
- Enable the checkbox "Set additional beam shift"
- Center the beam
- Disable checkbox "Set additional beam shift"
- Relative beam shift between Record and View is now set
Repeat the above procedure for "Focus"
If Focus and Trial are not identical, repeat once more for Trial
check by cycling that beam stays centered

Defining Focus/ Trial Area

Take a "View" picture
Change checkbox "Define position of Area" to "Focus"
On View image, click the area where you want Focus to be
- Focus area will always be along tilt axis, so you are simply defining the distance along this axis
- If you want it very far away (ie completely off of the View screen), type in the distance in microns (and hit Enter)
When done, set "Define position of area" to none
Re-check centering of Focus beam, and change value of "Set additional beam shift" if necessary
If Focus and Trial are not identical, repeat above procedure for Trial mode

Automation and SerialEM

SerialEM now allows more powerful automation, as Macros (and tilt series) can be run at a set of defined points
To take advantage of this, need to use the Navigator within SerialEM for map-making
Once a map is made (entire grid or part of a grid), points of interest can be added:
- One by one -- choose "Add points", and manually click on the map. When done, click "Stop Adding"
  - New points will have to be marked "Acquire". Highlight point and enable "Acquire" checkbox, or type "a"
- A grid of points (2.88):
  - In the map, choose "Add polygon", and add points in the map which define a closed polygon
  - Under Navigator, choose to "define a grid". Choose the desired separation of grid points (default = 1um, which is very close)
  - Can then choose to automatically activate all points, instead of hitting "A" 100 times
Once a set of points are marked as Active, choose "Acquire at areas" under Navigator menu
Can now choose to collect a tilt series at each area, run a macro, or take an image
- Some example macros are on our Macro page
Good idea to check "Close column valves when done", especially if you intend to let it run for hours

The Navigator

Navigator part of SerialEM is used to make maps (montages) of grid areas
Points of interest can be added to maps, then marked for tomogram collection, automatic image collection, etc
In general, first step is to set up a low-mag montage of entire grid, then make higher-mag maps of interesting areas

Making a map of the entire grid

first, open the Navigator:
Set microscope to 170-200X mag, objective aperture out, reasonable brightness
Setup Record image to high binning (4 or 8), and short exposure (0.1s)
Take a test Record image with these settings
In Navigator Menu, choose to setup a full montage
Defaults fine in montage menu, but increase the overlap to 15%
For saving the file, choose to save everything in extended header. All other defaults are fine
- Use the file extension ".st" for stack
Montage control panel will appear
- Uncheck "create sloppy montage" and click "Start" to start the data collection
Montage will take ~ 20 minutes
When done, choose to make a new map

Making higher mag maps

One way to make a submap at higher mag is to do a ploygon montage
set up the microscope to higher mag (~1700X, or a low step in mag mode)
In Navigator main screen, choose to Add Polygon
- Left click to add points for polygon, then click "Done adding" when done
Select the polygon and choose Montage/super montage-->create Polygon montage
- Make the map, and again choose to make new map from this montage

Aligning the low-mag map with higher mag maps

Alignment between low-mag mode and normal mags may not be perfect, so the following will align the whole-grid montage with higher mags
Select a higher-mag montage which has some recognizable feature, and choose "Change Registration"
- Make the higher mag map registration 2
On the low-mag map, pick 3-5 points which you can also recognize on the higher-mag map.
Mark these points as Registration points

Mark the corresponding points in map 2 as Registration points, being sure they are numbered the same (1 corresponds with 1, etc)

Be sure that the Current Registration is 2 before going on to the next step
In the Navigator Menu, choose "Transfrom Items"
If everything is OK, all items will now be in registration 2
If points do not correspond well, SerialEM will warn you and you can go back to re-pick or move points

Automatically adding a grid of points for acquisition

From Polygon

On a map, draw a polygon
Under Navigator menu, choose to Add a Grid of points
Choose desired separation, and a grid will be added within this polygon

From a visible grid (eq. Quantifoil)

On a map image, Add 5 points which define the grid
- 3 corners, plus 2 points adjacent to one corner
Select one of the points, and choose Navigator-->Add grid of points
When dialogue comes up, just hit OK
Points will be added to match grid

Using SerialEM search mode rather than Tecnai Low dose

latest version of SerialEM has a new mode, "search" at left edge of low-dose control panel
search mode is ONLY for searching -- there is no associates camera mode
set up as follows

Go into Tecnai low-dose as normal.
Go to exposure mode. You will stay there your entire session.
Set up View, Focus, Record, Trial as normal.
Choose "Search" as area to show when screen down.
Take a "View" picture.
Lower screen.
Go to diffraction mode. Choose spot size 9, D 1.35 M (screen is down).
Check and uncheck continuous update of mag and beam (to save these settings).
If beam is not in center, diffraction needs to be aligned as follows.
1. Be sure Continuous Update of Mag and Beam is unchecked.
2. Go to Tecnai Tune, under Image HM-TEM choose Align Diffraction Pattern.
3. Your settings will change. Raise then lower screen using Screen lift on microscope (NOT R1).
4. Using Multifunction X-Y, move beam over approximate position of off-axis camera. May need to lower distance to Trash.find DFdf beam.
5. Raise screen, move toggle to TEM mode, and unblank beam using SerialEM unblank button.
6. Using multifunction X-Y knobs, center feature of interest to match "view" picture.
7. Note that Fastscan camera settings should be "No Mirror" "Rotate right 90" to match SerialEM.
8. Click "Done" under Tecnai alignment when finished.

Instructions for using this mode

To go to search, be sure that "Search" radio button is highlighted as mode to go to when screen is down
Lower screen.
Switch toggle to TEM.
Raise Screen.
Unblank beam.
To go back to view, switch toggle to CEM and take a view picture.

Using SerialEM search mode rather than JEOL MDS

As with Tecnai, latest version of SerialEM understands diffraction mode and has a Search area in low-dose mode
Set up is as follows:
Set up View and Record as you normally would.
If you want to do a live focus rather than let SerialEM do it, set up focus as follows:
1. Select Focus under Area to show when screen down, then lower screen.
2. Change magnification to ~200K, adjust brightness and spot size as desired.
3. Check then uncheck Continuous update of mag and beam to save above parameters.
4. Check Additional beam shift, then move beam over off-axis camera using JEOL beam shift knobs.
5. uncheck Additional beam shift to store this value.
6. Take a View picture, and Select Focus under Define Position of Area.
7. Left-click somewhere on the View screen where you want Focus to be. It will always be along tilt axis.
8. Select None under Define position of area.
Set up SerialEM Search mode as follows:
1. Take a View picture.
2. Click "search" under Area to show when screen down.
3. Lower screen.
4. Set spot size to 5, set mag to 10k, expand beam.
5. Check then uncheck Continuous update of mag and beam to save settings
  - NOTE JEOL version does not remember diffraction mode as of version 2.74.
6. Change JEOL to SA DIFF mode.
7. Move FLC panel CL1 all the way to the right.
8. Move beam over off-axis camera using PLA shift.
9. Raise screen.
10. Move toggle to TEM (from CCD).
11. Click Unblank Beam on SerialEM low dose control panel. You should see an image on live camera.
12. Use PLA shift to finely adjust position of live image to match the picture you took in View.

Instructions for using this mode

To go to Search, make sure that Search radio button is selected under Area to show when screen down.
Be sure that BLANK beam when screen down is selected.
Drag FLC Panel CL1 all the way to the right.
Lower screen, press SA DIFF, raise screen, move toggle to TEM, and click Unblank beam
You should now see a live image.
Before taking any View or exposure pictures, be sure to:
1. Move toggle back to CCD.
2. Click "All Off" on FLC control panel.
To go to a live Focus view, instructions same as for search except no fiddling with FLC panel (keep off) or SA DIFF mode.

Visit the Cryo EM Website at http://www.nysbc.org/facilities/CEM

-- DavidStokes - 28 Jul 2005