An Interactive User Interface for Automated Acquisition of Transmission Electron Micrographs

For several years we have been developing a software application, called Leginon, for the automated control and acquisition of images from a transmission electron microscope ^1,2,3. The system has been developed around a Philips CM200 and a Gatan MSC CCD camera. One of the primary motivations in developing this software is to provide for a system that can acquire many hundreds of images under low dose conditions from a specimen embedded in vitreous ice. In order to set up and manage this system we have developed a number of simple interactive graphical tools that enable the user to design, oversee and manage protocols for controlling the microscope and collecting the images. Many of these simple tools have also proved generally useful as stand alone applications.

The Leginon system performs a number of tasks automatically once a collection protocol has been defined by the operator. The automated software systematically scans the grid starting with the collection of a low magnification image of selected grid squares. The low magnification image is analyzed to identify target holes that contain ice of suitable thickness and specimens of appropriate density and distribution. An image of each target hole is acquired at an intermediate magnification to assess the quality of the specimen within the hole and to define an appropriate high magnification imaging strategy. Procedures for focusing and adjusting astigmatism, as well as ensuring that the specimen is not drifting, are then performed under low dose conditions. Finally a high magnification image is acquired.

The user interface has been developed using the Tcl/Tk scripting language ⁴ interfaced to the viewit image processing package. The interface communicates to the TEM using Tcl extensions to the emScope software library ¹. The main interface (figure 1) provides overall control of the experiment and information on the current status. It allows the user to specify how images should be saved and analyzed and whether the results of any procedures should be manually confirmed. It can also be used to launch a number of subsidiary stand alone tools, including an interface to set up the low dose kit and one to manage the selection of grid squares (figure 2).

The Leginon low dose setup interface allows for the creation of various preset imaging conditions similar to the low dose mode buttons (search, focus, exposure) on the TEM console. An unlimited number of presets may be recorded and saved. For each of these preset conditions we also record specifications for the CCD camera configuration used to record the image. These include exposure time, size, and binning factor. In addition, if the camera sensitivity has been calibrated, the electron dose for each preset is measured and recorded.

The selection of grid squares to be systematically examined is managed using the grid window (figure 2). The user is required to perform a simple calibration that records the orientation and size of the grid squares and the interface then displays a map of the grid squares that are accessible within the range of the goniometer. The current imaging position is indicated on this map and the operator can navigate around the grid using the mouse. The interface is also used to specify a protocol for automatically scanning the grid. The default option is to systematically spiral out from the current position. The operator can also specify particular paths or individual grid squares. Areas that the operator wishes to avoid and those that have already been imaged by the Leginon software are crossed off.

All options and parameters displayed on the control windows can be modified interactively while data acquisition is in progress. If the process is quit, or the system crashes unexpectedly, the interface can be restarted and the system will be restored, allowing the process to start up exactly where it left off. The system can also be monitored using a web browser interface that displays the current status of the experiment and the last set of acquired images. If the system needs attention or problems are encountered, this information is automatically sent to the operator's e-mail account and optionally to an alphanumeric pager.

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