Why do additional frames appear in SymPhoTime when opening a multi-frame image acquired in NIS-Elements?
When a multi-frame FLIM image acquired in NIS-Elements is opened in SymPhoTime (SPT) using a multiframe analysis script, two additional frames may appear.
For example, an image recorded with 10 frames in NIS-Elements may appear as 12 frames in SymPhoTime. This behavior is expected and originates from the acquisition process rather than from SymPhoTime itself.
What causes the extra frames?
The additional frames arise due to how the scanner hardware operates:
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First frame (pre-scan frame):
The AX (and A1) scanners perform a pre-scan before the first valid frame is acquired.
This frame often contains only partial lines and is not suitable for analysis.
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Last frame (incomplete termination):
The acquisition does not always stop exactly at the intended number of frames.
As a result, the final frame may be incomplete or contain residual data.
The same behavior can occur with other stop conditions as well.
Can the first pre-scan frame be removed automatically in SymPhoTime?
Yes. SymPhoTime provides a setting to automatically exclude the first frame from FLIM and FRET analyses.
To enable this:
Start SymPhoTime in Configure Mode
Click Settings → Edit factory settings
Open ImagingAnalysis
Set the current setting to True
Click OK
Go to Settings → Save hardware configuration
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Enter the serial number and your name, then click OK
After this configuration, the first frame will be automatically excluded from all new FLIM and FLIM-FRET analyses.
What about the last frame?
The last frame is not removed automatically.
If it is incomplete or not required, it can be excluded manually by entering the appropriate “last frame” number in the multiframe analysis script.
Does this affect the actual measurement data?
No. The additional frames are acquisition-related artifacts and do not indicate an error in the measurement itself. Proper configuration and frame selection ensure that only valid data are included in the analysis.