4.8 Special Topics: Increasing Area for ‘Supermontage’
Reconstructions
4.8.1 Using SerialEM to Acquire Supermontage Tilt Series
Single or dual axis tilt series are acquired from large area domains of each section,
using tools within the Navigator control and the user interface of the SerialEM
image acquisition software. The Navigator allows one to set up super-montage
maps with appropriate overlap, from which montage tilt series are automatically
acquired (Fig. 4.7a, b). It is important to name the montaged tilt series from the
individual panels with information in the file name that refers to the position of the
panel in the supermontage. For example, the panel in Fig. 4.7b would have a name
ending in _x2y1z1.mrc to indicate that this panel of the supermontage is the right
most panel in x and from the first section in z.
Steps to set up a supermontage tilt series acquisition using an example of a
2 Â 1 supermontage of 3 Â 3 montages.
1. Insert grid, adjust eucentricity
2. Find cell/area of interest and pre-irradiate entire cell to *2000 e-/A
2
3. Create an overview map at medium magnification (*4700Â) by opening a
new montage, typically 2 Â 2 pieces. Check ‘stage montage’ and acquire.
4. Open a Navigator window and make the image a new map.
5. Go to the desired tilt series magnification and open a montage file with 3 Â 3
pieces.
6. Double click on the overview map line in the Navigator table to read in the
map. Using the left mouse button, click in the center of the image area that you
want to acquire.
7. From the top SerialEM menu, select Navigator—Montaging & Grids—Setup
Supermontage and define as a 2 Â 1 supermontage.
8. Turn on Acquire for the new entries in the Navigator table
9. From the top SerialEM menu, select Navigator—Acquire at Points, checking
Realign and Acquire map image options in the popup window. Hit go.
10. Setup beam autocentering.
11. In the main Navigator window, highlight the line of the first acquire map and
check Tilt Series. This will bring up a file properties window. Select a montage,
set it up as 3 Â 3, name the file and set the increment and total angular range.
12. Select the other lines as Tilt series. The files will automatically be named
sequentially but final names can be set at this time by the operator.
13. From the top SerialEM menu, select Navigator—Acquire at Points.
– Choose Autocenter beam, Realign to item, Close column valves at the end
of the tilt series and Send email, Acquire tilt series.
108
E. O’Toole et al.
Reconstructions
4.8.1 Using SerialEM to Acquire Supermontage Tilt Series
Single or dual axis tilt series are acquired from large area domains of each section,
using tools within the Navigator control and the user interface of the SerialEM
image acquisition software. The Navigator allows one to set up super-montage
maps with appropriate overlap, from which montage tilt series are automatically
acquired (Fig. 4.7a, b). It is important to name the montaged tilt series from the
individual panels with information in the file name that refers to the position of the
panel in the supermontage. For example, the panel in Fig. 4.7b would have a name
ending in _x2y1z1.mrc to indicate that this panel of the supermontage is the right
most panel in x and from the first section in z.
Steps to set up a supermontage tilt series acquisition using an example of a
2 Â 1 supermontage of 3 Â 3 montages.
1. Insert grid, adjust eucentricity
2. Find cell/area of interest and pre-irradiate entire cell to *2000 e-/A
2
3. Create an overview map at medium magnification (*4700Â) by opening a
new montage, typically 2 Â 2 pieces. Check ‘stage montage’ and acquire.
4. Open a Navigator window and make the image a new map.
5. Go to the desired tilt series magnification and open a montage file with 3 Â 3
pieces.
6. Double click on the overview map line in the Navigator table to read in the
map. Using the left mouse button, click in the center of the image area that you
want to acquire.
7. From the top SerialEM menu, select Navigator—Montaging & Grids—Setup
Supermontage and define as a 2 Â 1 supermontage.
8. Turn on Acquire for the new entries in the Navigator table
9. From the top SerialEM menu, select Navigator—Acquire at Points, checking
Realign and Acquire map image options in the popup window. Hit go.
10. Setup beam autocentering.
11. In the main Navigator window, highlight the line of the first acquire map and
check Tilt Series. This will bring up a file properties window. Select a montage,
set it up as 3 Â 3, name the file and set the increment and total angular range.
12. Select the other lines as Tilt series. The files will automatically be named
sequentially but final names can be set at this time by the operator.
13. From the top SerialEM menu, select Navigator—Acquire at Points.
– Choose Autocenter beam, Realign to item, Close column valves at the end
of the tilt series and Send email, Acquire tilt series.
108
E. O’Toole et al.