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hci-unihd / antibodies-analysis-issues

Issue tracker for problems in the antibodies analysis workflow.
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Using well H01 for background - plateK12rep1_20200430_155932_313 - H01-0004 #60

Open tischi opened 4 years ago

tischi commented 4 years ago

{"plateName":"plateK12rep1_20200430_155932_313","siteName":"H01-0004","pixelLocation":[1659.0545454545456,22964.42181818183,0.0],"analysisVersion":"f917e50cb8cce34d0481d6d7d3ee3c5e2a7baffa"}

@imagirom @metavibor

I am looking more at this well that could be used for background measurments.

Observations:

  1. Background value is around 1300 for IgG.
  2. The average measured background corrected control cell intensity in IgG is -300 in this well. This shows that the plate_median was too high for this well. I guess this indicates that the sticking to the coverslip of the serum is an effect that happens in the majority of the wells (and thus further motivates to potentially rather use this well for the background measurement).
  3. The flatfield correction is still not perfect, one clearly sees a shape in the signal. Maybe could still be improved? If I measure the background at the edges I get values around 1240.

Screenshot

metavibor commented 4 years ago

@tischi @imagirom

Background value is around 1300 for IgG.

I measured H1 well in several plates and I'm getting a mean value of 1400 +/- 50 across different plates.

The average measured background corrected control cell intensity in IgG is -300 in this well.

yes. I think this is more of a problem than I initially thought. We should take the H1 value in the future

The flatfield correction is still not perfect, one clearly sees a shape in the signal. Maybe could still be improved? If I measure the background at the edges I get values around 1240.

I think the first thing to do is to perform the flatfield correction and than measure the background. What d you think? Regarding improving the correction - I don't know, I think @imagirom has done some averaging approach initially and found a correction pattern that might be better than what I obtained with the chroma slide. We could try that. Alternatively, the background pattern in H1 well after some Gaussian blurring could be used for correction

tischi commented 4 years ago

I think the first thing to do is to improve the flatfield correction...

I agree with this, let's first try to get this as good as possible, because the flatter the field, the better we can do background correction later.