Energy Filtering

SerialEM allows convenient use of an energy filter (post-column or omega filter) and efficient tilt series acquisition with filtering. For Thermo/FEI scopes, it relies on the EFTEM software being installed.  On JEOL scopes, it works with an omega filter or with a post-column GIF, where it recognizes the GIF mode and may be able to control it. It provides a number of additional features when used with a post-column filter.

• Automatic switching between regular lens mode and EFTEM or GIF lens mode when the screen is raised or lowered. For the occasions when you need to see the constricted beam of EFTEM lens mode on the screen, you just toggle this automatic switching off.
• Automatic switching between upper camera and EF camera.
• Automatic changing of magnification to provide the least change between the pixel sizes on the upper camera and on the EF camera.
• Automatic intensity adjustment so that the electrons per pixel per second will be the same on the EF camera as on the upper camera.
• Integration with low dose mode so that different filter settings can be used for pictures of the different low dose areas.

Even without SerialEM, there are numerous ways to control filter settings: the EFTEM panel in the Thermo/FEI Microscope User Interface (MUI), the Autofilter palette in DigitalMicrograph, and the Filter Control program in GMS 1 or its equivalent dialog in DM in GMS 2-3. SerialEM tries to be a good citizen among this crowd and respond to changes made in other interfaces. The recommendation from Thermo/FEI was (and may still be) that the filter be controlled only through the MUI. This is probably sound advice for any actions that cannot be done through SerialEM. Specifically, this includes aligning the zero loss peak (ZLP), tuning the EF, and performing spectroscopy.

The biggest problem in using the EF for tilt series is that there may be an energy shift when changing to lower magnifications for various alignment tasks. If the slit is in, the zero loss peak may shift out of the energy range of the slit, and the task will fail. One solution would be to remove the slit when going to lower magnification, but this would cause an unpredictable and potentially large increase in intensity that could saturate the camera. The solution in SerialEM is to calibrate the energy shifts and apply these automatically when changing magnifications, so that the slit can be left in and will still be centered at the lower magnification. The program will also apply energy offsets to accommodate changes in slit width on older GIFs. These capabilities allow you to explore different magnifications and slit widths without having to align the ZLP repeatedly. repeatedly.

SerialEM implements the three standard imaging modes: unfiltered (with the slit out), filtered to record only zero-loss electrons, and filtered to record electrons with a specific range of energy losses. Once filtering is selected, it is possible to switch between zero-loss and a specific energy loss simply by toggling the 'Zero loss' checkbox; however, if you want a different slit width in the two cases you need to adjust it each time you switch between them. See Filter Control panel for more details.

The Filter Control Panel in SerialEM includes a button to refine the alignment of the ZLP. The procedure is referred to as a refinement because it will work only if the ZLP is already close to aligned, say within 10 eV. The Align ZLP function available in other interfaces must be used for initial alignment, because it is capable of finding the ZLP from many eV away. However, the refine ZLP procedure will align the ZLP even when it is a very small fraction of the total electrons, because it concentrates on the drop in signal as the slit is moved off of the left edge of the ZLP. Thus, once a thick specimen is in, the refine ZLP procedure may be more reliable than the Align ZLP procedure run from elsewhere.

Steps for Using a GIF on a Thermo/FEI Scope

1. Be sure to start Filter Control (for GMS 1), then DigitalMicrograph, then SerialEM after DigitalMicrograph reports Dynamic cameras registered in its Results window.
2. Go to a mid-range magnification like 20,500 with the screen down. Adjust the beam to a spot size and screen current that will get you through the tuning procedure without having to adjust intensity. (Specific values vary widely; if you have to turn the beam down in the First Order isochromaticity adjustment, put the screen down when you get through the tuning and note the current for the next time.)
3. Activate EFTEM mode in the Filter Control panel and take a picture through the GIF just to verify function.
4. Set the slit width to 10 to 20 ev; 20 seems preferable.
5. Press 'Align ZLP' on the Thermo/FEI EFTEM panel.
6. Press 'Tune GIF' on the Thermo/FEI EFTEM panel.
7. Switch to the magnification that you will be using for final image acquisition. Lower the screen and adjust the beam to fill the screen with enough intensity for a good gain reference (5 - 10 na?).
8. Raise the screen and take a gain reference.
9. Now you should assess the adequacy of the calibration of mag-dependent energy shifts (unless you will be working in low dose mode and having the slit out for pictures of the View area). To do this:

• Set Trial parameters for a fast picture with moderately high counts, e.g. 256 x 256, center half area, 0.05 - 0.1 second exposre.
• Turn on intensity zoom.
• Set slit to 15 ev.
• Go to a low mag like 5600. Take pictures with the slit in and the slit out and measure the mean counts (Ctrl-I).
• Repeat for other low mags that may be used in tasks (e.g. 4500 and 3000).
• If you see some serious beam loss or dark spots on the edges with the slit in, you need to calibrate the mag-dependent shifts, as described in Mag Energy Shifts . Enter the highest mag that you will use and accept the defaults for the other parameters.