SerialEM Note: Touch Up Low-Dose Offsets for IS and Defocus

Author:: Chen Xu
Contact:: <chen.xu@umassmed.edu>
Date_Created:: May 19, 2026
Last_Updated:: May 19, 2026

Abstract

There are still a couple of issues in the Low Dose setup that occasionally require attention or correction. For example, in our experience, after running final data collection for some time (e.g., around 20 minutes), the Image Shift offset for View area can drift slightly. As a result, the target position may become off by about 100 nm or so. This is not a major issue, but it can be annoying when very accurate targeting is required. We have observed this behavior not only on Talos and Glacios, but also on Krios. I suspect this is related to a hysteresis effect. It would be useful to have a way to correct this quickly, or even automatically, during data collection.

Another issue can occur after an unexpected exit, such as a program crash. After restarting the SerialEM program and entering Low Dose mode again, there may be a so-called “defocus doubling” problem in View or Search mode. For example, if View mode was originally set with a -200 µm defocus offset, it may become -400 µm after restarting. It would be helpful to have a simple way to fix this issue as well.

In this note, I will share how I handle these problems.

To Fix IS Offset

There is a script command available to obtain a new, hopefully good Image Shift offset for View and Search. Here we are focused on View.

FindLowDoseShiftOffset #S [#M] [#S] [#A]

This works equivalently as [Auto] button in Low Dose Control panel. It is important to make sure there is a strong feature in both Record and View mags so the above command can work reliably. Lets say there is a black ice chunk somewhere we can use. Here are the steps to prepare:

1. Take a Preview image contain this black chunk or edge of the chunk. Make it into a map. Assume it has index # 3.

2. Take an anchor map at View mag. This is to help to realign accurately to the Preview map, specially when Preview mag is high.

And I can create a little script as below”

ScriptName Fix-IS-Offset

RealignToOtherItem 3 0           # index = 3
FindLowDoseShiftOffset 0         # 0 is for View Offset

You can run this little script whenever you like to. And you can also run this periodically in your collection workflow pipeline.

To Fix Defocus Doubling Issue

When using the latest Testing executable, we sometimes encounter a “defocus doubling” issue after a crash. Specifically, after restarting the SerialEM program and turning on Low Dose mode, the microscope defocus may be at -400 µm, even though the Low Dose defocus offset for View mode is set to -200 µm.

The root cause of this problem is that View mode uses the uP probe mode, while F, T, and R typically use the nP probe mode. If SerialEM crashes while in View mode, the microscope may be left in uP mode with a defocus of -200 µm, while the nP mode defocus remains at 0. One simple way to fix this is to press the Eucentric Focus button on the right-hand panel before restarting SerialEM.

For a normal-exit crash, the latest Testing version of SerialEM can detect the crash upon restarting and prompt you with an option to fix the issue automatically.

However, if you do not notice that the microscope is already at -200 µm defocus and you start SerialEM and enter Low Dose mode without correcting it first, then View mode will inherit the existing -200 µm defocus in uP mode and apply an additional -200 µm offset from the Low Dose settings. As a result, the View defocus becomes -400 µm.

This can be easily fixed by a fairly simple script as below:

ScriptName Fix-Defocus-Doubling
ToolbarFillColor gold

# fix double defocus offset problem due to crash

# turn option off
ReportUserSetting AdjustFocusForProbe
If $RepVal1 == 1
   SetUserSetting AdjustFocusForProbe 0 1
Endif

# LD on
ReportLowDose
If $repVal1 == 1
   Echo In LD!
Else
   Echo Now turn it on!
   SetLowDoseMode 1
EndIf

# fix it
GoToLowDoseArea R
SetStandardFocus 0
GoToLowDoseArea V
SetLDDefocusOffset V 0
SetStandardFocus 0
SetLDDefocusOffset V -200
SetUserSetting AdjustFocusForProbe 1 1

# ReportLDDefocus
ReportLDDefocusOffset V