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Normalizer causes gaps?

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mayanlevy

Participant

Posts: 9

Joined: Thu May 11, 2017 10:26 am

Post Sat Feb 06, 2021 2:23 pm

Normalizer causes gaps?

Hello!
I seem to have a problem with the Helios normalizer and would love some help.
After using the normalizer, there are sudden gaps in the lower parts of every channel such as those (ignore the blue square):
Image
I tried to increase the time interval from 100 to 120 and then 150 but it doesn't seem to work.
We also tried slightly increasing the amount of beads we put in the samples, but that didn't work either.
What are we doing wrong?
Any help would be appreciated.

P.S. This image is taken from the MATLAB normalizer, but the normalization was actually done on Helios

Thank you,
Mayan
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kiyolega

Participant

Posts: 2

Joined: Wed Mar 24, 2021 7:28 am

Post Thu Mar 25, 2021 12:25 am

Re: Normalizer causes gaps?

I recommend searching with cytof randomization.
viewtopic.php?f=3&t=1655

kiyota
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Verhoeff

Participant

Posts: 19

Joined: Wed Mar 02, 2016 10:25 am

Post Thu Apr 29, 2021 8:55 am

Re: Normalizer causes gaps?

Hi Mayan,

It has to do with the randomization function in the FCS processing. By default it is checked, so it should randomize the integer output of the TOF to a more visually pleasing spread around that integer. Maybe it has been disabled by mistake.

Or possibility is a bug in the software. We've encountered it a few times where users simultaneously normalize an FCS file through the FCS processing, and acquire a sample. The increased workload for the workstation can cause a bug where the randomization step is skipped. Rebooting the software and then re-processing the FCS files solves the issue for us.


Jan

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