SerialEM Note: An Alternative Way to Center a Hole
- Author:
Chen Xu
- Contact:
- Date_Created:
June 8, 2025
- Last_Updated:
Jan. 22, 2026
- Abstract
If the current stage position is slightly off from the center of a hole and you want to center it, a common approach is to use a hole template for alignment.
However, there is an alternative method. The hole-finding routine in SerialEM is both powerful and robust. You can detect hole positions and center on the closest one without using AutoAlign and a reference image. This can be done easily with a script command.
In this note, I will share how I use this function, along with two examples.
Two Examples
Fig.1 Center Hole Before and After
The image on the left shows a typical LD_View image at 2250× magnification in uP mode on the Krios. The center of the hole marked with a green marker indicates the desired position for automatic centering.
In this case, I can simple run a script command as below:
FindAndCenterOneHole 0 1.3 0 2
This will center the hole that is closest to the center of camera, as shown on the right. The green marker is for visual indication only—the procedure itself does not rely on it. Instead, it centers the nearest hole using Image Shift.
The command utilizes the Hole Finder function. A prerequisite is that you have already run the Hole Finder from the dialog window on this grid. In this command, 1.3 specifies the hole diameter, or -1 can be used for the value stored in user setting file or 0 for stored in map; all other parameters are inherited internally from the dialog settings.
While this method works even if there is only one hole in the area, it’s not limited to such cases. When applied to regions with many holes, it may run slightly slower—but even then, the delay is minimal. For a single hole, the operation typically takes around 0.1 seconds. If the field of view contains many holes, you can save time by cropping the center area, as shown in the function in script below.
This can be easily expanded to a more general usage, as shown in below script:
ScriptName CenterHole
## a script to quickly move to center of a hole. If no marker point,
## it moves the hole that is closest to the center of the image. If there
## is a marker, it will move to the center of the hole that is the closest to
## marker point.
## define for cropping and hole size
useCrop = 1 # 0 if not use crop
buf = A # A buffer
size = 5 # 5 um
holeSize = 0 # -1 from setting, 0 from map or actual diameter in microns.
### not editing below ######################
ImageMarkerPosition 0 X Y
If $X == -1 AND $Y == -1 # no marker present
If $useCrop == 1
CallFunction CropCenterMicron $size $buf # uncomment to crop
Endif
Else
MoveToMarker
V
If $useCrop == 1
CallFunction CropCenterMicron $size $buf # uncomment to crop
Endif
Endif
FindAndCenterOneHole 0 $holeSize 0 2
ClearHoleFinder
V
##
Function CropCenterMicron 1 1
ImageProperties $buf X Y Bin Exp pixelSize
size = $size / $pixelSize * 1000
size = ROUND $size 1
CropCenterToSize $buf $size $size
EndFunction
That is, when there is no marker present in the image in current buffer, the script above will center the closest hole to the 0,0 of the image. If there is a marker present, it will center the hole closest to the marker point. You don’t have to put marker exactly in the center of that hole. This can very useful to move around to holes, for example, for a quick screening purpose.
As you can see, this approach provides an effective alternative to hole template matching method. During actual data collection or screening, you can use just a few lines of script to accurately center on each hole across the grid.
# RealignToNavItem 0 # use Realign, ended with a V shot
MoveToNavItem # or just move to the item
V # need a shot
FindAndCenterOneHole 0 -1 0 2
ClearHoleFinder
ShiftItemsByAlignment # continously update the X Y of items, prevent wandering away
If Realign routine is used, there will be an image from it, so there is no need to take another LD_View shot. If you use MoveToNavItem instead, you will need to take a shot for hole finder to use.
This method can also be applied within the StepTo & Adjust dialog to refine Image Shift (IS) vectors for a multiple-exposure pattern. See the image below:
Fig.2 Centering Hole to refine IS vectors
As shown, I used 15kX as the intermediate magnification to refine the Image Shift (IS) vectors. At this magnification, a single hole fits within the field of view. With Automatic Adjustment turned on, the system moves to each corner hole, centers it, and sets the corresponding IS value.
In my experience, the IS determination at this magnification is sufficiently accurate for use at high magnifications such as LD_R at 130kX. However, you can further refine it by using the hole edge as a landmark after running the auto-adjustment at the lower magnification (15kX here).
As of June 8, 2025, the FindAndCenterOneHole command is available in versions 4.2 and 4.3 testing branches, with a few bug fixes. It was initially introduced in 2024.
To Center to the invisible hole in the middle of 4 holes
When multishot pattern with even number like 2 x 2, 4 x 4, etc. are used, precsiely centering to the center of 4-hole pattern is challenging, as there is no center hole to use. How to center to this invisible middle hole?
Fig.3 Centering to invisible hole of middle of 4
The task here is to go to center of the “invisible” hole in the middle of 4 holes, indicated by the green marker point.
Recent (23-Sept-2025) implemented command can help with this task. The command is
ReportSmallestHoleShift 0
Together with the center hole command described above
FindAndCenterOneHole 0 1.3 0 2
This task can be accomplished by a script like below:
ScriptName ToMiddle4Holes
## obtain multishot pattern, if not available yet.
#FineHoles 0
#UseHoleVectorsForMulti 0
## hole size - 0: from map, -1: fromm setting or actual size (1.89)
holeSize = 0
## Shift to closest corner hole of the multishot pattern
ReportSmallestHoleShift 0 ISX ISY
ImageShiftByUnits $ISX $ISY
## center the corner hole of 4
V
FindAndCenterOneHole 0 $holeSize 0 2
ReportImageShift ISX1 ISY1
## shift back, twice distance to hit the opposite hole
ImageShiftByUnits ( -2 * $ISX ) ( -2 * $ISY )
## center to the opposite corner hole of 4
V
FindAndCenterOneHole 0 $holeSize 0 2
ReportImageShift ISX2 ISY2
## obtain middle IS values and shift to there
finalISX = ( $ISX1 + $ISX2 ) / 2
finalISY = ( $ISY1 + $ISY2 ) / 2
SetImageShift $finalISX $finalISY
ClearHoleFinder
V
The idea is to utilize the multishot pattern available, shift to one of the corners, center it and then shift to opposite corner hole and center it again. Thus, the middle of the two corner holes can be obtained and it shifts to there. Only image shift (IS) is invloved here.