Automated EM Data Acquisition with SerialEM

Download and Installation Page
Tilt Series Acquisition with SerialEM
Single Particle Acquisition with SerialEM
Program Features
Contribute to the Continuation of SerialEM
Highlights of SerialEM 4.1
Availability of SerialEM for Various Hardware Configurations
Setting Up SerialEM
How to Report a Problem with SerialEM
SerialEM Discussion Group
Other Helpful Materials
Source Code for SerialEM and SerialEMCCD Plugin
Mirror Site for SerialEM

SerialEM is a program that can acquire a variety of data from electron microscopes: tilt series for electron tomography, large image areas for 3-D reconstruction from serial sections, and images for reconstruction of macromolecules by single-particle methods.

The main focus of the program is automated tilt series acquisition. For this, it uses an approach based on prediction of specimen position during the tilt series from the position at previous tilts. It does not count on the microscope or the specimen being particularly well-behaved, so unless a prediction appears reliable, it falls back to measuring and adjusting defocus and/or specimen position when necessary. With this method, it achieves both robustness and speed. For example, a 2Kx2K, 1 degree tilt series can be acquired in about 20-25 minutes with a four-port readout CCD camera.

The method and the program are described in this paper:
Mastronarde, D.N. 2005. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152:36-51.
The prediction method was first described in this abstract from the 2003 MSA meeting,
Mastronarde, D.N. 2003. SerialEM: A program for automated tilt series acquisition on Tecnai microscopes using prediction of specimen position. Microscopy and Microanalysis Vol. 9, Suppl. 2, 1182CD.

SerialEM is also widely used to acquire data for single particle reconstruction. After positioning on a hole, it can acquire multiple image in a hole and/or in multiple holes using image shift, with compensation for the beam tilt induced by the image shift. There is a routine that finds hole positions automatically and one that can combine them into an optimal subset of positions for acquiring from multiple holes. The basic framework for this automated acquisition is through the Navigator module, which in SerialEM 4.0 includes an enhanced interface for acquiring from the selected points and performing all commonly associated actions.

In addition to automating data acquisition, the program provides an integrated user interface for image acquisition, display, and storage. It looks like this:

Click image for full-sized view.

Other notable features include:

• The program provides a flexible interface for user intervention. When acquiring a tilt series, the user can stop if necessary, adjust position or focus, and even conveniently back up to a previous tilt angle and replace the stored data from that point onward. Other kinds of acquisition also provide an ability to stop and restart.


• There is a low dose mode, in which focusing and tracking are done in a separate area to minimize exposure of the area being recorded. The program allows one to set up the magnification and illumination independently for up to 4 different areas, where these settings are invoked simply by taking a picture of the particular area.


• SerialEM can automatically acquire a montage of overlapping frames, and it can acquire a tilt series of such montaged images. The images are written to a single file that can be viewed immediately in 3dmod. A montaged tilt series can be processed with the Etomo interface in IMOD.


• SerialEM can also acquire energy filtered images through a Gatan Imaging Filter (GIF), a JEOL omega filter, or a Thermo Fisher Selectris. Both low dose mode and montaging can be used with the filtering.


• The program supports imaging and tilt series acquisition in STEM mode.


• There is a Navigator window that allows one to store and return to specimen positions, make low-magnification maps, mark positions on maps, return to marked positions reliably, and automatically acquire images, tilt series, or montages at a series of positions.


• The program has a scripting capability that includes variables, 1D and 2D arrays, simple arithmetic statements, looping, and conditional statements. It is most commonly used to acquiring images automatically for single-particle reconstruction; some scripts are available for this purpose. The script capability also provides a relatively easy way to add commands requested by users for specialized or experimental use.


• The operation and configuration of SerialEM is completely documented in a Windows compiled HTML help file opened from the program. You can access the help online in HTML format for either the current stable release version or the development version of SerialEM. In addition, you can download and view the compiled help file for the development version after unzipping on any Windows machine. The on-line help and compiled file for the development version are continually updated and will often show features that are not in the current release version.

SerialEM was originally developed with grant funding from NIH. Since 2014, it has been maintained and developed with funding that comes from users, which has allowed us to focus on coding and documenting instead of grant-writing. Since 2018, funds have also been used to pay Guenter Resch to provide user support.

You can help make SerialEM a community-supported resource by having your laboratory or microscope facility contribute to the further development and continued availability of the program. The mechanism for doing this is to purchase priority annual support, so that the expense represents a tangible item that can be paid from grant or institutional funds. In September 2020, we transitioned to having these payments made to Guenter Resch's company, Nexperion. In addition to directly funding Guenter's assistance to users, The majority of the funds are used to compensate David Mastronarde for his work on SerialEM, with the rest directly funding assistance to users by Guenter and by Chen Xu (at the University of Massachusetts Chan Medical School). We offer three options, allowing you to pick the size of your payment based on your financial resources and the importance of SerialEM to your work. The options are:

1. Basic support: Priority resolution of issues arising from SerialEM bugs and consultation of issues arising from configuration and calibration problems ($600 non-profit, $1200 commercial).
2. Software support: In addition to basic support, advice and consultation on how to optimally use the workflows available via SerialEM’s user interface ($1200 non-profit, $2400 commercial).
3. Application support: In addition to software support, advice and consultation on how to get the most out of SerialEM for complex applications, e.g. via scripting ($2400 non-profit, $4800 commercial)

What happens if you do not pay? Although the first option represents the kind of support that we provide routinely, payment for this support is voluntary. We do answer questions and deal with problems from non-paying users, but may take longer to do so.

Why is it important to contribute? User contributions are important both financially and because they are not tied to specific developments and represent the general user interest.

What does the change in 2020 mean for the future of SerialEM? This is not a move toward commercialization. We intend to keep the program freely available. It is already open source.

How do I get help with SerialEM under the new arrangement? For everyone, requests for support and for initial configuration (framework) files should still be sent to serialemsupport@colorado.edu. (See How to Report a Problem with SerialEM).

Click Here to go to the registration page for support agreements on Nexperion's web site. (This initial registration is required only once.)

The most notable features of the latest release, SerialEM 4.1, are:

• Autocontouring of grid squares, with a dialog to set various selection criteria for which contours to convert to Navigator polygons
• Improvements in montage alignment for periodic specimens and whole grid maps
• Support for hole finding, combining and acquisition on gold grids with a hexagonal array of holes, including removal of periodic signal from cross-correlations
• Improvements in refining image shift vectors for Multiple Records and deriving the vectors using hole positions from Hole Finder
• New task in Navigator Acquire at Items to run Hole Finder and Combiner after making a map
• Saving of shifts from Shift to Marker operation
• Inclusion of Ctfplotter from IMOD in the package and integration into the program as an alternative to the ctffind module, to get improved CTF fitting on lower dose and tilted images
• Expansion of imaging states to include Low Dose Focus and Trial
• Extension of capabilities for running external programs
• Ability to display graphs if IMOD or a standalone 3dmod package is available
• Addition of 118 script commands
• Upgrade from 2008 to 2015 compilation tools

There are many other improvements; see the list of notable features to learn about them.

SerialEM works on modern Thermo Fisher (FEI) microscopes (Tecnai, Titan, Spirit, Talos, Glacios), JEOL microscopes with the TEMCON or TemCenter interface (e.g., 1400, 2100, 2200, 2500, 2800, 3100, 3200, ARM200, F200, CryoARM), and the Hitachi HT7700 and HT7800. The program is built and tested primarily with a Tecnai simulator on Windows 10, but it still works on Tecnais under Windows 2000.

SerialEM works with CCD or CMOS cameras from Gatan, TVIPS (Tietz Video and Image Processing Systems), Thermo Fisher, AMT, DirectElectron, and EMSIS (formerly OSIS, Olympus Soft Imaging Solutions); it also supports the JEOL Ruby and other cameras. Direct detection cameras from Gatan, Thermo Fisher, and DirectElectron are supported. The EIGER2-based EM detectors from DECTRIS are also supported through a plugin currently available from DECTRIS. All EMSIS cameras work through a plugin available from EMSIS for either 32- or 64-bit versions. STEM acquisition is supported from Gatan's DigiScan, Thermo Fisher's STEM imaging, the TVIPS scan generator, and the JEOL STEM on the 1400 series, F200, CryoARM, and possibly other systems with similar interfaces.

If necessary or advantageous, SerialEM can be run on a separate computer and control the microscope over the network. Such a configuration is standard on JEOL microscopes, is quite common when using DE and newer Gatan and Tietz cameras on Thermo Fisher microscopes, and is also possible for Hitachi microscopes. The program can access Gatan, Direct Electron, Thermo Fisher, and Tietz, and JEOL cameras over the network from another computer, but for other kinds of cameras, SerialEM must run on the computer that hosts the camera.

For running on a Thermo Fisher microscope, it is necessary to have the Thermo Fisher scripting adapter installed. If you do not have the adapter, you need to purchase it from Thermo Fisher. To check whether it is installed, do the following:

1. Locate and run the "Delphi Scripting Exampler". For Tecnais through version 3 and possibly on older Titans, this is available through the Start menu under Programs - Tecnai. For Tecnai version 4 and some newer Titan software, it is under Programs - FEI Company - TEM Software. On Windows 10 systems, it is under Thermo Scientific.
2. Select the "Projection" tab after the program starts.
3. Press the arrow next to "Magnification". If a magnification appears, you have the adapter; if not, there will be an error message in the status bar at the bottom of the window.

Although SerialEM itself is open source, the microscope plugins and some camera plugins in this software are not. The University of Colorado retains various rights with regard to these components; however, they are currently provided free for both commercial and non-commercial purposes. By downloading the distributed software, you agree that you will not distribute it outside of your own institution.

The program has been installed on over 400 Thermo Fisher and 200 JEOL microscopes.

Program and configuration files are available on the Download and Installation page. Follow the instructions in there to get started. As a first step, fill out the questionnaire in Word doc form and send it to serialemsupport@colorado.edu; we will prepare an initial configuration file. This is strongly recommended for TFS/FEI scopes and absolutely essential for JEOL and Hitachi scopes. Installations on new JEOL microscopes are usually done with the assistance of Jaap Brink from JEOL (jbrink at jeol dot com), but sending in the questionnaire is still the starting point.

Guenter Resch from Nexperion offers SerialEM installation and training for both Thermo Fisher and JEOL microscopes. This service is available worldwide, on site or remotely. Chen Xu at the University of Massachusetts Chan Medical School and Gabriel Frank at Ben Gurion University in Israel may also be willing to help with installation; email

for contact information.

Send all problem reports and requests to serialemsupport@colorado.edu. This is a private email address, not a mailing list. Guenter Resch is the primary contact for getting assistance with SerialEM. He will collect needed information about a problem, and handle most problems with use of the program or pass them on to Chen Xu. Problems in the program itself will be handled by David Mastronarde.

What we can support: We can only diagnose problems that occur in relatively recent releases. If you are running a version prior to the current stable release version (e.g., 3.8.10, or a 4.0 beta version, after 4.0 is in stable release and 4.0.x packages are available), you must upgrade and see if the problem still occurs. If you are running the current stable release version but it is not the latest update (e.g., you have 4.0.5 and the latest is 4.0.12), you should upgrade before reporting the problem. If you are running a beta version in the current development cycle that is not based on the latest beta release package, upgrade if possible, or at least check the History-beta.txt or History-testing.txt file before reporting the problem.

The following information and files can help to address your question faster and more effectively. Here are the essentials:

• The exact wording of the error message
• A saved log if it might contain relevant information, especially the log after a crash. When the program crashes, it saves to the log file if one was defined, otherwise to a file SerialEMcrash.log (or SerialEMcrash1.log, etc), typically in the directory where the initial settings file was located.
• Screenshots of error messages, SerialEM, and/or the microscope user interface. Please send a real screenshot of the whole SerialEM window and associated components. We find that cell phone pictures are often unreadable, and that some useful information is often just outside the range of a partial screenshot.
• A precise description of steps that lead to the issue you are reporting
• How often the problem occurs (every time, sometimes, about once a day)
• The type of microscope and its location (city, institution, and laboratory).
• The type of camera being used when the problem occurred.
• The exact version of SerialEM, including whether it is 32- or 64-bit, and for a beta version, the build date. This information appears at the start of the log when the program starts, and as of SerialEM 3.7 it is in the crash message.

These additional points can also be helpful:

• With SerialEM 4.0.2 or higher, run the command "RunSerialEMSnapshot" in a script window or in the one-line script window. Save the log and send this log. If you are not able to run this command, send SerialEM properties and calibrations (SerialEMproperties.txt and SerialEMcalibrations.txt, typically found in C:\ProgramData\SerialEM or C:\Program Files\SerialEM). Also describe which directory the files came from.
• If the properties file is not available when you report, describe the layout of your system (what hardware is used, what computers are present, where is SerialEM installed)
• If you are not able to run the SerialEM snapshot, provide the settings file that you were running from. (If not a specially named file, right-click the shortcut that you run SerialEM with, select properties and find SerialEMsettings.txt in the "Start in" folder shown there.)
• The version of the operating system, including whether it is 32- or 64-bit, and of the microscope user interface being used
• Whether you tried any of the relevant troubleshooting procedures described in the SerialEM help, and if so which ones.
• If the program hangs, for how long did you let it sit before killing it?

Please follow these additional tips for effective communication:

• Do not reply to a previous message from the SerialEM list or one of us; start a fresh message with its own title line.
• Report one issue per email unless there is some apparent relationship between the issues.
• Follow this terminology for the behavior:
  • Crash: the program closes, generally after an uninformative error message from SerialEM and/or Windows.
  • Hang: the program becomes unresponsive and has to be closed with Task Manager.
  • Error: a procedure stops with an error message from SerialEM
  • Bad state: the program interface is responsive but many operations are grayed out and pushing STOP does not restore it.

The SerialEM discussion group provides a means for users and people managing the program to ask questions and share their experiences, as well as a way to receive notification of new releases and other news. Digests and posting via email (i.e. list server functionality) are provided. Additional capabilities, including browsable / searchable archives are available through a web interface. Access to the web interface requires a Google login profile, which must be associated with the email address at which you wish to be contacted. (Non Gmail addresses can be used with a Google login, with or without a Gmail address. See the Email section of your Google Account settings). The postings are not moderated, but joining requires approval.

To subscribe or visit the group via the web interface please visit SerialEM. If you do not have and are not willing to create a Google login and are satisfied with email only access, you can also request membership by sending an email to

asking to be added to the SerialEM list, and you will be added manually. Be sure that this email is sent from the same address as you would use to post to the list.

After your request is approved, you will be added to the group and will receive a confirmation email with additional instructions. Once subscribed, we request that you edit your membership settings for this group so that your display name is your real name. To post to the forum you can either use the web interface or simply email your posting to serialem@colorado.edu

To cancel your subscription, use the web interface or send an email to serialem+unsubscribe@colorado.edu.

When replying to a previous posting via email, you will have the option to reply to the sender or to the list.

A standalone copy of 3dmod and associated files that can be run on Windows without installing IMOD or Cygwin is available here. The package unpacks to C:\Program Files\3dmod by default and a shortcut can be set up to run 3dmod from there. The package also includes midas, which can be useful to have on the microscope for checking rotations between magnifications. The latest package includes ctfplotter for determining defocus and astigmatism, and genhstplt for displaying graphs, and SerialEM 4.1 includes commands to use these.

An optional plugin to SerialEM can provide direct access to JEOL microscope functions through the TemExt interface without relying on Python. It enhances SerialEM scripting capabilities and allows for an event-driven scripting approach. It is available here. See the README.txt file for requirements.

A repository for contributed scripts was developed by Guenter Resch from Nexperion and is hosted on the Nexperion site. If you have scripts that may be useful to others, please consider adding them to the repository.

Guenter has also developed a web page on the Nexperion site that can prepare a Basic mode file for a particular set of activities, so that controls not relevant to those activities can be hidden.

Daniel Caujolle-Bert at the University of Basel has written a program for managing user and settings and scripts in an environment where multiple users share the same login; it is available here.

Guenter is creating a new set of video tutorials on particular operations; so far they include autocontouring grid squares and backing up a SerialEM installation. These are available on our Vimeo page.

Our YouTube channel has a series of videos of the lectures on SerialEM presented in a webinar in September 2012, plus some tutorial videos prepared by Cindi Schwartz. The videos are also available right here; if you sort on the "Last modified" column they will be in a good order for viewing.

Here are some training documents written by Cindi Schwartz, to provide concrete guidance for doing room temperature and cryo tomography.

• Optimizing Plastic Section Tomography
• Making Navigator Maps and Setting up Low Dose
• Strategies for Cryoelectron Tomography

Here are Powerpoint presentations from a workshop on SerialEM given in December 2006. The slides have a lot of words, but there are many fewer words than in the help manual, so they may be helpful by highlighting the most important points.

• Introduction to SerialEM
• Low dose, Navigator, Energy Filtering
• Scripts, Camera issues, Setting up the program

And again, here are the:

• Help in HTML format - stable release version
• Help in HTML format - development version
• Windows compiled HTML help file - development version

Both SerialEM and the SerialEMCCD plugin to DigitalMicrograph are open source; SerialEM under an MIT license, and SerialEMCCD under the GPL version 2. You can browse the SerialEM source here and SerialEMCCD source here, or you can download either repository with Mercurial. See the IMOD source instructions for setting up Mercurial and updating a repository. This SerialEM repository has been pruned of all changes before Feb. 17, 2017 so as not to disclose calls to proprietary microscope and camera interfaces. It is now used for development, so those who wish to contribute changes can send a mercurial bundle from this repository and no longer need access to the older repository.

To clone the SerialEM repository, run

    hg clone http://bio3d.colorado.edu/SerialEM/SerialEM

    hg clone --insecure https://bio3d.colorado.edu/SerialEM/SerialEM

To clone the SerialEMCCD source, run

    hg clone http://bio3d.colorado.edu/SerialEM/SerialEMCCD

If you are going to try to compile either of these projects, you will also need the collection of libraries, which is now in a separate SerialEMLibs repository. You can download this with:

    hg clone http://bio3d.colorado.edu/SerialEM/SerialEMLibs

The module for Python support is also available here. You can browse the project files here or you can clone this repository with:

    hg clone http://bio3d.colorado.edu/SerialEM/PythonModule

Occasionally, this site becomes inaccessible for various reasons. There is a mirror site available at https://bio3d-mirror.nexperion.net