Montage Usage Notes

05-02-06 updated May 2008

Collect gain reference before beginning
- take a record image of the vacuum (binning 2, 1 sec exposure)
- Enter record counts into Target counts box (cntl+I will give you the counts in the log file window)
- select binning of 2

* Low mag mode - 170X (150X with screen down)

Spot size 3 beam completely spread
Montage images taken in Record mode (0.1 s exposure time)
Open Navigator file
Use set up full montage from navigator pull down (Do not use montage set up from file menu)
Use 10% overlap (11x11)
save as bytes and .st file
save Navigator file as .nav
Good idea to keep tracking on during lowest-mag montage in order to view progress
Tilt axis is ~100-110 degrees in the onscreen ccd images
Good idea to empty buffer tank before starting montage, otherwise it may start during acquisition

After setting up exposure level and magnification, take a test image. Note minumim/maximum densities. This will be used to set levels later.
Choose to "Align Pieces in Overview" in Montage pane. Also choose to view montage when done.
Open the Navigator window.
For subset of grid
- Mark 4 points to make a large rectangle to cover most of grid
- Mark each point as a Corner Point using checkbox
- Navigator Menu - Setup Corner Montage
For whole grid
- Navigator Menu - Setup Full Montage
- Note - at 220K, binning 4, full montage is 16X14 images.
Choose "byte" rather than integer to save memory space. Bytes have range 0-255, so it asks range for scaling. Get range from values in test image.
Choose a reasonable filename.
Go over to Montage pane (left bottom of screen), click start. Be sure you are ok - you can't stop it once it is going.
Come back in ~30 minutes for a full grid.
Once done, click "New Map" in Navigator window. You should now have a montage of the whole grid.

Next steps: Find interesting ares of grid for higher-mag views. However, the 2 magnifications must be aligned as follows

Increase magnification to 2500 or 3500X. May have to adjust brightness, spot size to keep dose low.
Find an interesting area of the grid, with some recognizable features.
Choose "Add Polygon", draw a polygon to map out this interesting area. Make it larger than 1 square, since alignment is likely far off.
After polygon is drawn, mark it as "Acquire" with checkbox.
Under Navigator menu, choose "Setup Polygon Montage". Again, choose bytes. May need another test image to estimate levels.
Once set up, click "Start" on Montage panel.
Important - After done, increase Register level from 1 to 2. Otherwise you can't do alignment.
Now that register has been changed, click "New Map" in Navigator Window
You now have 2 maps: Low-mag with Register 1, higher-mag with Register 2.
Compare high mag and low mag montages to Trash.findDFdf an equivalent point. The alignment may be off by 2 or 3 gridsquares.
To make this easier, do a screen capture of high mag image (shift-printscreen), paste into MS Paint application
Change register back to 1, load low-mag map, (map 1), choose add point, and add a point on the feature.
Mark it as a "Registration Point" using checkbox in Navigator window.
Increase Registration to 2, load map 2 (high mag map), add a point on the matching feature, mark it as a registration point.
Go to Navigator Window, choose Transform. This will transform map 1 (low mag) onto map 2.
Re-load low-mag map. Note that it is now listed as Map 2, since it has been aligned.
Choose "Add Polygon", and draw a polygon to map out a grid square (any square)
Choose "Setup Polygon Montage" under Navigator menu.
Find areas of the grid you are interested in, and add points to the middle of these squares.
Mark all these points as "Acquire" using checkbox in Navigator window
Under Navigator Menu, choose "Acquire Areas". This will acquire polygons around all your points.
Once you Trash.findDFdf good areas to image, you can go to them directly using SerialEM.

-- BillRice - 02 May 2006