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Acceptable / typical background levels

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jimbomahoney

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Posts: 45

Joined: Wed Feb 27, 2019 11:21 am

Post Wed Jul 31, 2019 8:52 am

Re: Acceptable / typical background levels

EDIT - The below was conjecture / misunderstanding on my part, but I'll leave the post as-is.
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Another thought that I had after discussing with Fluidigm was the LCT (lower convolution threshold).

The default is 400 (DCs I assume).

Therefore, this corresponds to solution mode as follows:

(1000/13) * 400 = ~31k DCs.

This is because one push is 13 µs, whereas the solution mode acquisition is an "accumulation" over 1 second.

Therefore, if contaminants are present higher than 31k DCs in solution mode, this may impact the detection of weak events by pushing the LCT up and hiding them.

Thoughts / comments appreciated as always!
Last edited by jimbomahoney on Thu Aug 01, 2019 8:23 am, edited 1 time in total.
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jul 31, 2019 3:30 pm

Re: Acceptable / typical background levels

Hi James,

I'm not sure that's completely correct.

The LCT is a summary across *all* channels. This is one reason why you may want to increase the LCT for high-parameter experiments or decrease it for low-parameter experiments. Even in a well-stained sample, each channel will have a certain amount of background, which can add up.

Unless you're having streaking in all or most channels or *terrible* background in just one, I haven't seen much issue with cell event determination (ie, Event Rate going really high as it thinks every bit of debris is an event, or even worse, Event Rate drops really low as it starts thinking you have *REALLY LONG* cells since the between-event data never drops below the LCT). I *think* this is because you usually have at least a few really bright markers (such as Ir) that helps drive the true Events' signal well above the LCT.

If anything, I've had at *least* as many problems with event rates and resolution when users run their samples too fast (even if the sample is "clean").


Mike
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jimbomahoney

Contributor

Posts: 45

Joined: Wed Feb 27, 2019 11:21 am

Post Thu Aug 01, 2019 8:22 am

Re: Acceptable / typical background levels

Thanks Mike.

Yes, I chatted again with Fluidigm, who explained that the LCT units are not pulses or DCs - it's more of an internal / software unit, so it's not as simple as I had understood it to be.

And yes, I understand now that the LCT is getting pushed up by Ir anyway. I'll edit my original post too.
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