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Hi,
I have been experimenting with the different programs for Li's ICQ method
to optimize my analysis. I find that the ICA plugin for ImageJ gives ICQ
values that do not correspond to the PDM image when I select thresholding.
I have used a control with staining of the same protein using two different
fluorophores (GFP and antibody) which appears to give all positive PDMs
based on the PDM image however the ICQ is very low (ICQ of -0.4 with
thresholding and 0.4 without).
In an attempt to avoid this problem I switched to Fiji's Coloc2, which
appeared to fixed this problem for the control. However based on figures
that should have low ICQs they seem to be inflated. When I select smaller
ROIs the ICQ goes down, so it seems like zero-zero pixels are being taken
into account despite thresholding? I use cells so it is difficult to avoid
zero-zero pixels as we move up the focal plane there is an increase in the
number of zero-zero pixels. I tried solving this by using a mask, but
values are lower than would be expected (ICQ = 0.2 for control used above).
I was wondering if anyone has encountered this problem or knows of a
solution.
Much appreciated,
Alexandra Sokolovski
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