Acquiring Multiple Tilt Series
Multiple tilt series can be run in a batch mode by using the Navigator to define
positions where the series are to be taken, setting the parameters for the
acquisition in advance as the positions are selected for tilt series in the
Navigator, then starting the tilt series from the Navigator
Acquire dialog box .
Points to consider: There are various considerations for using
Each target position should be located within a medium-magnification Navigator
map that is large enough to allow the position to be found reliably with the
Realign to Item command. Such a map is referred to as an anchor map,
and Navigator has features to simplify taking such maps. It is recommended that
you also take a map at the magnification for the tilt series, and corresponding
to a zero-degree view of the target area, as this map will allow more precise
positioning, but this map is not essential.
You will probably want to use the beam autocentering task to protect against
the beam position wandering during protracted data collection. To do so, you
need to set up the task to work properly at the magnification where you are
acquiring data, using the Beam Autocenter Setup
dialog box . Once this is set up, you should test the task before data
collection to see if its parameters need adjustment (run the
Autocenter Beam command in the Tasks menu or use the Shift-E hot key).
Cooking plastic sections.
If you are working with plastic sections, you can make the pre-exposure of the
specimen be part of the batch operations. To do so, you need to set up the
specimen cooking task using the Specimen Cooking Setup
dialog box . Once this is set up, you can run that dialog to check the
parameters before data collection and make sure that they are appropriate for
the extent of area being acquired.
Bidirectional series. For reliable results, it may be necessary to
take the tilt series in two directions from 0 tilt. This will usually be
the case with cryo-samples, where the walk-up to a high starting angle or
tracking down from it may fail. It may also be desirable to use a
bidirectional series with other samples where there is difficulty focusing at
high tilt, since it is generally easier to stay in focus as high tilt is
approached in small steps from below than to establish good focus when starting
the series at high tilt.
Finding tilt range.
Often you need to set the tilt angle range separately for the individual tilt
series, after assessing what range is feasible at each location. You may find
the Tilt Series Range Finder helpful for
assessing the tilt range. If you use it and then select angles at the end of
the range in the Tilt Series Range Viewing dialog,
then you need to open the relevant tilt series parameters (using the TS
Parameters button in the Navigator) to have the selected angles become the
limiting angles for that series. Note that as of SerialEM 3.4.0, a batch
tilt series is considered to be successful if an image that is too dim is
encountered at a high enough angle, as specified by settings in the Tilt
Series Batch Policies dialog. Thus, you may be able to skip assessing the
ending angle. Since the same criteria are applied to terminate the first
part of a bidirectional series upon a dim image, you may be able to skip
assessing the tilt range completely for such series.
Eucentricity and Z height.
As always, the tilt series will run much more smoothly if the eucentric height
is set for the specific location. Moreover, if the specimen is going to be
pre-exposed, then the final eucentricity setting should be done after cooking.
This can be achieved by selecting to refine eucentricity either in the
Navigator Acquire dialog box or the Tilt Series Setup dialog. However,
this routine cannot run reliably unless the Z height is within ~10 microns of
eucentric. There are two options for taking care of this: select the Rough
Eucentricity task in the Navigator Acquire dialog box,
or set the eucentricity approximately by whatever means you prefer when you set
up the series at each point. If you set the Z height after adding the Navigator
item that is marked for a tilt series, select the item and change its Z height
with the Update Z button in the Navigator.
You need to make a basic choice of what should happen in case of an error:
whether the tilt series should stop with a message or whether the Navigator
should terminate it and go on to the next. The Tilt
Series Batch Policies dialog has an option for this basic choice and
some other options relevant to running tilt series or other automated
operations. These include an option to have a separate log file for each
series, and an option to control whether and when to close column/gun valves if
a message box is open for a long time.
Receiving email notification can be very helpful when running multiple series,
especially if you select for a message box come up when there is an error. In such
cases, an email will be sent before the box is opened. To send mail, the
program needs to be able to contact an SMTP server and this server needs to be
defined in the properties file. Once this has been configured, you will be able
to enter an email address in the Tilt Series menu and select the option to have
For each tilt series, you have the option to store an imaging state consisting
of scope settings such as beam intensity (or, in Low Dose mode, the entire set
of parameters for the record area), and Record parameters such as exposure
time. In Low Dose mode, the axis position of the Focus area and an offset from
center of a subarea selected in the Focus camera parameters is also stored. This capability is obviously useful if you want a different state for
some tilt series, or a different Focus area in Low Dose mode; it can also be used for the first tilt series to guarantee
that a particular state is set then.
Stopping and intervening: When running tilt series from the
Navigator, it is still possible to intervene manually, but the situation is
somewhat complex because of the two layers of control involved. There are three
distinct phases where the End and STOP buttons in the Camera and Script control
panel have three different effects.
During the actions run by the Navigator prior to the start of the tilt series,
such as rough eucentricity, the STOP button will stop the action and stop the
Navigator. The current item will still be marked for tilt series acquisition,
and you can resume from the Navigator Acquire dialog
box . The End button (labeled End Nav) will signal the Navigator to end
after this tilt series is finished.
During the startup phase of the tilt series, before the first image of the
series is acquired, the STOP button will end the current operation and put the
TSC into a 'postponed' state. When resumed from this state, the TSC will go
through the whole sequence of startup operations again. Pushing the Resume
button will open the Tilt Series Setup dialog box
, from which you can restart the series. Alternatively, you can abandon this
location and go on to the next tilt series by terminating the series, either
from the Tilt Series menu or by pushing the End button (now labeled End TS).
If you want to end all acquisitions, then select 'End Acquire' in the Navigator
Menu BEFORE terminating the tilt series. During the startup phase, pressing the
End button (labeled End Loop) will cause the TSC to stop after the saving the
first image of the series.
Once the tilt series is through the startup phase, STOP stops the current
operation and End (labeled End Loop) stops it at the end of a loop. At this
point everything works as in a manually started tilt series. The Resume and
Back Up buttons function as usual, opening their respective dialogs. The
Tilt Series Setup dialog box can be opened to adjust parameters and
resume from there. The tilt series can be terminated from the Tilt Series menu
or with the End button (labeled End TS). Again, if you want to end all
acquisitions, first select 'End Acquire' in the Navigator Menu.
Step-by-step Procedures: There a several ways to use the various
tools provided for setting up multiple tilt series; here is one variation.
Make whatever maps are needed to locate your areas of interest. This could be
just a low-mag map, or a low-mag map plus a series of medium-mag maps. The
steps below for making anchor maps would not be needed if all points are
located in medium-mag maps.
Open a file for maps of the areas to be acquired.
Locate the first area of interest. Run the Rough Eucentricity procedure or
otherwise adjust the Z height to be approximately the eucentric height.
Go to the magnification required for the tilt series and acquire an image of
exactly the area to be imaged.
Save this image and make it a map.
Go to a magnification where the camera field of view is 10-15 µm (if you have a
state set up for this in the Imaging State dialog, set this state there).
Acquire an image and save it as a map.
Check 'For anchor state' in the Navigator dialog.
Return to the working magnification (use 'Restore Prior State' in the Imaging
Run the Tilt Series Range Finding procedure or otherwise determine the limits
of the series.
Select the first map again and check 'Tilt series' in the Navigator.
Define file properties as needed then define tilt series properties.
Go to the next area of interest, and adjust the eucentric height approximately
Acquire an image of the area to be acquired.
Press the Anchor Map button. This will save the first image as a map and
make a second map for an anchor.
Determine the angular range for the series.
Select the higher mag map again if necessary and check 'Tilt series'. This time
no dialogs will open.
Press the TS Parameters button to import an angular range determined
with the Range Finder and to adjust any other parameters
To adjust the automatically generated filename, press the Filename button.
Continue with steps 13 to 19 for any other areas to be acquired.
Check that beam autocentering is working correctly, if you plan to use it.
If appropriate, check the parameters for specimen cooking.
Select 'Acquire at Points' from the Navigator menu. Tilt series acquisition
will be selected as the action to perform.
Select 'Autocenter beam' if you are set up to use it;
'Realign to Item' to align to the maps at each position; 'Cook specimen' if
appropriate; and 'Fine Eucentricity if this operation is not selected in the
tilt series parameters for each series
Set 'Close valves at end' and 'Send email' as desired, then press the GO
The order of the steps can be varied. If you adjust eucentric height after
making a map item, you can press the Update Z button in the Navigator to
change the Z value of the map item. If you run the Range Finding procedure
after setting other tilt series parameters, press TS parameters to get
the new angular limits into the tilt series parameters.
If you are acquiring montaged tilt series, you may want to make the map at the
working magnification be a montage of the same size, although this is not
essential. In this case, you would have two files for the maps, one for these
montages and one for the anchor maps. A procedure for doing this would be as
Open a montage file for the maps at the working magnification.
Locate the first area for acquisition.
Press Prescan in the Montage Control panel to take a trial montage
Shift the prescan with the right mouse button and take another if necessary.
Take a full montage and make it a map.
Open another file for single frame images.
Go to a magnification appropriate for taking an anchor map.
Take an image and save it as a map. Mark this item 'For anchor maps'.
Return to the working magnification.
Select the montage file to be the current file, or just close the second,
Perform other tasks for setting up a tilt series at this location, as above
Locate the next area for acquisition.
Use Prescan to localize the desired area, then take a full montage.
Press Anchor Map.
Perform other tasks for setting up a tilt series at this location.
Repeat steps 13 to 15 for further areas to be acquired.
Note that the Anchor Map button becomes particularly convenient when maps
are kept in multiple files.