SerialEM Note: Accurate values for “Eucentric Focus” button
- Author:
Chen Xu
- Contact:
- Date_Created:
July 30, 2026
- Last_Updated:
July 30, 2026
- Abstract
It is quite common for the “Eucentric Focus” button on the right pad of the TFS microscope to set the focus inaccurately, especially at lower magnifications. One possible reason is that it is difficult to determine the true focus of a low-magnification image during the microscope alignment procedure.
In this note, I describe how SerialEM’s Autofocus function can be used to refine the value associated with the “Eucentric Focus” button, allowing it to set the focus more accurately.
Background Information
Pressing the “Eucentric Focus” button sets the microscope focus to a predefined value for a specific combination of magnification, probe mode, and TEM mode. For example, the stored value may correspond to 910× magnification, µP probe mode, and EFTEM mode. These values are established during the microscope alignment procedure.
During the alignment, you are typically asked to use the multi-function keys to bring an image feature or the crossed beam to the center of the screen and then carefully focus the image. At this point, the Eucentric Focus value for that particular magnification and mode combination is stored.
In normal operation, you do not need to press the “Eucentric Focus” button for these values to be used. Whenever you switch to a magnification, the microscope automatically recalls the stored focus value for that magnification and mode combination and applies it to the objective lens. This is why the Obj% value usually changes when you change magnification.
At high magnifications, it is relatively easy to achieve accurate focus during the alignment procedure because image features are clearly visible. At low magnifications, however, image features are often difficult to see well enough to judge the exact focus. You can use a direct camera to assist with focusing, and continuous imaging with Thon ring information can also help. Even so, it is still possible to store a focus value that is off by 50–100 µm, because it is difficult to determine the true zero-defocus point from Thon rings at low magnifications.
Although this level of error is usually not critical for routine operation, it can be inconvenient. Each time you switch to a low magnification, you may find that the focus is approximately 100 µm away from the desired value and need to correct it manually. It would therefore be beneficial to refine the stored Eucentric Focus values so that they are more accurate.
SerialEM Autofocus Comes to Help
You can use SerialEM to refine these values.
Assuming that Image Shift has been calibrated for all magnifications and Autofocus has been calibrated for at least one magnification in the SA range, you can run the following SerialEM script to determine the “true” focus value for each magnification, especially in the low-magnification ranges (such as M and several SA magnifications). The script performs autofocus at zero target defocus and prints the corresponding objective lens value to the log.
SuppressReports
SetTargetDefocus 0
Autofocus
ReportObjectiveStrength OBJ
obj = round $OBJ * 100 4
Echo Obj = $obj%
SuppressReports 0
ReportMag
Echo
After recording the objective values for each magnification, repeat the corresponding microscope alignment procedure. Instead of relying solely on visual focusing, manually adjust the focus knob until the objective lens value matches the value determined by the SerialEM script. Then complete the alignment procedure so that the Eucentric Focus value for that magnification is stored.
By repeating this process for the magnifications of interest, you can significantly improve the accuracy of the stored Eucentric Focus values. As a result, switching to those magnifications will place the microscope much closer to the correct focus, reducing or eliminating the need for manual focus adjustments during routine operation.