Diffusing molecules cause a negative step amount

[NicolaasvanderVoort]

Dear Johan,

It was very nice meeting you at the picoquant conference. As promised I report an issue we encounter when measuring in cells.

We measure in cells on membranes. Sometimes we also get unwanted diffusion of fluorescent species in our data. When this happens at the end of a trace, the number of fluorophores becomes negative. I've attached an example of an analysed graph and the binned data.

Trace.txt

It is a problem because in my analysis I'd like to assume the number of steps is positive, it is also an unphysical result. As a workaround I exclude these traces for further analysis. As a desired solution, I suppose that changing the counting such that the lowest #fluorophores is always 0 would solve the issue. We can post-filter to exclude traces that do not have 0 fluorophores at the end of the trace.

Please let me know if you need additional information.

Thanks in advance!

[JohnDieSchere]

Hi Nikolaas

I also liked meeting you at the workshop and am always happy to see people use my Code. I think I understand the problem and it should be easy enough to fix... I'll let you know when I have a workable solution on the git. For the time being filtering those traces out is probably a good preliminary solution.

best wishes Johan

Am Mo., 11. Okt. 2021 um 12:48 Uhr schrieb NicolaasvanderVoort < @.***>:

Dear Johan,

It was very nice meeting you at the picoquant conference. As promised I report an issue we encounter when measuring in cells.

We measure in cells on membranes. Sometimes we also get unwanted diffusion of fluorescent species in our data. When this happens at the end of a trace, the number of fluorophores becomes negative. I've attached an example of an analysed graph and the binned data.

Trace.txt https://github.com/JohnDieSchere/quickpbsa/files/7322124/Trace.txt [image: AnalyzedTrace] https://user-images.githubusercontent.com/49991393/136785064-71a21ee3-f28b-46bc-b2e9-f6752d51dc18.png

It is a problem because in my analysis I'd like to assume the number of steps is positive, it is also an unphysical result. As a workaround I exclude these traces for further analysis. As a desired solution, I suppose that changing the counting such that the lowest #fluorophores is always 0 would solve the issue. We can post-filter to exclude traces that do not have 0 fluorophores at the end of the trace.

Please let me know if you need additional information.

Thanks in advance!

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[JohnDieSchere]

Hi Nikolaas

Sorry for the delay, I only started working on this now... But now I realize I don't quite understand what's going on. At first I thought the diffusing molecules would cause the mean signal to be higher for the background than for the fluorophore , but your mean is actually lower... In that case the fluorophore number should not go negative. Would you mind sending me a few of the traces where this happens? I think that would make it much easier to find the issue.

best wishes Johan

Am Do., 14. Okt. 2021 um 17:11 Uhr schrieb Johan Hummert < @.***>:

Hi Nikolaas

I also liked meeting you at the workshop and am always happy to see people use my Code. I think I understand the problem and it should be easy enough to fix... I'll let you know when I have a workable solution on the git. For the time being filtering those traces out is probably a good preliminary solution.

best wishes Johan

Am Mo., 11. Okt. 2021 um 12:48 Uhr schrieb NicolaasvanderVoort < @.***>:

Dear Johan,

It was very nice meeting you at the picoquant conference. As promised I report an issue we encounter when measuring in cells.

We measure in cells on membranes. Sometimes we also get unwanted diffusion of fluorescent species in our data. When this happens at the end of a trace, the number of fluorophores becomes negative. I've attached an example of an analysed graph and the binned data.

Trace.txt https://github.com/JohnDieSchere/quickpbsa/files/7322124/Trace.txt [image: AnalyzedTrace] https://user-images.githubusercontent.com/49991393/136785064-71a21ee3-f28b-46bc-b2e9-f6752d51dc18.png

It is a problem because in my analysis I'd like to assume the number of steps is positive, it is also an unphysical result. As a workaround I exclude these traces for further analysis. As a desired solution, I suppose that changing the counting such that the lowest #fluorophores is always 0 would solve the issue. We can post-filter to exclude traces that do not have 0 fluorophores at the end of the trace.

Please let me know if you need additional information.

Thanks in advance!

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub https://github.com/JohnDieSchere/quickpbsa/issues/2, or unsubscribe https://github.com/notifications/unsubscribe-auth/APSQBAR2B57FQ322NSHJTNDUGLFKHANCNFSM5FYBM3QA . Triage notifications on the go with GitHub Mobile for iOS https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675 or Android https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub.