Introduction to SerialEM
SerialEM presents an integrated environment for image acquisition, display, and storage, as well as for the control of the microscope needed to acquire montaged images and tilt series. You can acquire images from a variety of cameras using parameters that you set from within SerialEM. Images can be zoomed, panned, and compared with each other using controls similar to those in Imod. Successive images are conveniently saved to a single MRC file, ready for display in Imod. The Tilt Series Controller will acquire a tilt series automatically using a prediction algorithm to minimize acquisition time and specimen exposure. A low dose mode can be used to perform focusing and tracking away from the area of interest. SerialEM can also automatically capture a montage of overlapping frames, and acquire a montaged tilt series. The program also contains a script feature for programming repetitive actions.
Controls: SerialEM is controlled through its menus, control panels, and hotkeys. Menus contain commands related to a topic, as well as some entries to set parameters related to that topic. The control panels provide status information, buttons for performing all frequently used operations, and buttons for setting parameters related to the particular panel. Here is some general information about Control Panels. Hotkeys are summarized in the section on Mouse and Keyboard Controls.
Buffers and display windows: Images are initially displayed in a main image display window. Images are kept in a set of buffers, and the main display window can show an image from any one of these buffers. Similarly to 3dmod in IMOD, you can riffle through the images with PageUp and PageDn. The buffers are referred to by letter. There are currently 20 buffers, A through T. Images from the camera are always placed in buffer A, the first buffer. The program is typically set up to roll images through the first three buffers. In other words, when a new image comes in, the existing image in buffer A is moved to buffer B, the image previously in buffer B is moved to buffer C, and the image in buffer C is lost. However, it is often desirable to increase the number of images kept in rolling buffers, which can be done with a spin button in the Options section of the Buffer Control panel. The display will show "Rolling" in the upper left corner when showing a rolling buffer.
It is also possible to copy an image to a new, free-standing window, which could be useful if you want to look at two images side by side.
The main display window will initially occupy the full area inside the program frame, and the program will resize it whenever you resize the program window. You can adjust the right and bottom edges of the window so that it takes up only a fraction of the area in the frame. This fraction will be maintained as the program window is resized, and it will be remembered between sessions of the program. The image window is kept in the top-left corner of the frame when the program adjusts the window size.
The lower left corner of the image will show the image values corresponding to black (0) and white (255) in the display. These are the same as the Black/White values in the Image Display control panel. The estimated image mean will also be shown.
Camera control: The camera controls are patterned after those originally available in DigitalMicrograph, but there are some important enhancements that provide more flexible control of the specimen exposure, make it easier to select subareas of the camera, and allow control of whether a dark reference is taken. General information about image capture is available in the section on Image Acquisition.
File storage: When images are saved to a file, successive images are typically stacked into a single file in the MRC format. The pixel size is set in the header, and tilt angle and other information can be stored in the extended header area or in a separate metadata file. The file header is maintained after every image is stored, so that the file will be readable if something goes wrong. Typically, a file is left open in the program so that multiple images can be saved or read back. There can be up to 100 open image files.
Image alignment: Alignment of images is a key component of data collection. SerialEM provides a linkage between the shifting of an image in a window, which is referred to as an alignment shift, and the physical realization of this shift using the image shift feature of the microscope. This means that if you want to center a particular feature in the camera frame, you can impose an alignment shift on an image that has already been acquired, simply by dragging it with the right mouse button. The microscope image shift is then changed by the right amount so that the next image that you acquire appears in the desired place. Two images can also be aligned to each other by cross-correlation. This is referred to as autoaligning. The image in buffer A is correlated to a reference image in another buffer. Before doing this correlation, the program will stretch the image taken at a higher tilt angle to give a better match to the other image. By default, the buffer after the last rolling buffer is used as the reference for autoaligning. When using the program's Low Dose mode, there are two default reference buffers instead: one for images centered on the area of interest (on-axis) and one for images taken at a position displaced along the tilt axis (off-axis).
Autofocusing: SerialEM determines the defocus of the specimen by the standard technique of measuring how much the image moves when the beam is tilted. When the specimen is in the focal plane, its image does not move; and the farther it is from focus, the bigger the beam-tilt induced movement. For this to work correctly, you need to align the beam tilt pivot points properly, a standard step in the Direct Alignments used to tune the microscope. Be sure that the specimen is at the eucentric height and at minimum contrast focus when you tune the pivot points. They do not need to be perfect, but do need to be close. Measurement of defocus is typically done by taking a picture with positive beam tilt, one with negative beam tilt, then another with positive beam tilt. Having three pictures allows the program to compensate for drift. Once the program has measured the defocus, it can change it to achieve the level of defocus that you specify. This procedure is called autofocusing.