Wobbler for Coma-free Alignment with Beam Tilt
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In some microscopes (e.g. FEI TEMs), the procedure of the coma-free alignment is very simple under the help of computer interface:
        i) Find an amorphous area on the specimen,
        ii) Click "Coma-free alignment x or y" on the interface of the microscope controller,
                The beam is wobbled in the x or y direction by tilting the electron beam back and forth by plus and minus ~10 mrad.
        iii) Adjust the beam tilt until the images for both tilts have the same apparent defocus, in other words, until the image that is seen with a plus tilt angle is indistinguishable from that seen with a minus tilt angle.

A procedure to obtain more accurate coma-free alignment is to generate tableaux of FFT patterns with a systematic beam tilt, similar to the method applied by Zemlin et al. [2]. As an example, Figure 2756 (A) shows a five-panel FFT tableau obtained from five TEM images after the alignment of the microscope but without coma-free alignment. The center panel shows the FFT pattern of the image obtained without any additional beam tilt, while the panels on the four corners show the FFT patterns of the images recorded after applying additional beam tilts of ± 5 mrad in the x and y directions, respectively. The beam direction in the central panel is not parallel to the coma-free axis, indicated by the fact that the astigmatism is slightly different for “plus” versus “minus” tilt angles. Therefore, a coma-free alignment was done by applying small beam tilts. After a small beam tilt, a tableau of FFT patterns similar to that presented in Figure 2756 (A) is recorded and compared. However, in most cases, the coma-free alignment cannot be completed by a single, small beam tilt, and thus this process is continued until the tableau is made as symmetric in appearance as possible. Figure 2756 (B) shows the FFT tableau after performing the entire process of coma-free alignment. In this tableau, the defocus and astigmatism for all the four corner panels are very similar and the tableau is symmetrical in both x and y directions.

Tableaux of FFT patterns obtained before (A) and after (B) coma-free alignments

Figure 2756. Tableaux of FFT patterns obtained before (A) and after (B) coma-free alignments. Adapted from [1]

 

 

 

[1] Robert M. Glaeser, Dieter Typke, Peter. Tiemeijer, James Pulokas, Anchi Cheng, Precise beam-tilt alignment and collimation are required to minimize the phase error associated with coma in high-resolution cryo-EM, Journal of Structural Biology 174 (2011) 1–10.
[2] Zemlin, F., Weiss, K., Schiske, P., Kunath, W., Herrmann, K.H., Coma-free alignment of high-resolution electron-microscopes with aid of optical diffractograms. Ultramicroscopy 3, (1978) 49–60.

 

 

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