Hi,
I would like to quantify and compare fluorescent intensities of
fluorescent in situ hybridizations (FISH) in two samples A and B.
The sample can be downloaded here:
sample A:
http://dl.dropbox.com/u/335344/sample_a.lsm (only the second
channel "Ch1-T2" is of interest)
sample B:
http://dl.dropbox.com/u/335344/sample_b.lsm (only the second
channel "Ch1-T2" is of interest)
Just by looking through the z-stack of the two samples it is obvious
that more cells are labelled in sample A than in sample B. Although I
have successfully applied the fabulous "3D object counter" plug-in to
nuclear stainings, I have been unable to delineate the cytoplasmatically
labelled cells in the above mentioned samples. Thus, the question is how
can I quantify the differences between sample A and B without counting
each cell individually?
I thought I could perhaps use the "measure stack" plugin to this end,
but I am not sure whether this is accurate or not too simple?. However,
if I run the plugin on the first 15 slides of sample A, I get:
Area IntDen RawIntDen
1 25613.91 205666817.63 8419539367
2 25613.91 205211843.31 8400913736
3 25613.91 204367338.41 8366341595
4 25613.91 205740033.99 8422536681
5 25613.91 205253167.18 8402605443
6 25613.91 200063364.07 8190146515
7 25613.91 195010541.89 7983295280
8 25613.91 193125786.33 7906137604
9 25613.91 193677959.79 7928742350
10 25613.91 193335915.28 7914739813
11 25613.91 189447340.58 7755550265
12 25613.91 183914692.74 7529056041
13 25613.91 181004949.43 7409937660
14 25613.91 179359183.58 7342563688
15 25613.91 178101664.69 7291083678
sample B:
Area IntDen RawIntDen
1 25613.91 79288698.12 3245901905
2 25613.91 77493149.48 3172396161
3 25613.91 76145067.04 3117208682
4 25613.91 74544633.14 3051690499
5 25613.91 73408611.51 3005184315
6 25613.91 72048328.91 2949497389
7 25613.91 70142404.70 2871473116
8 25613.91 67655541.54 2769666502
9 25613.91 65503786.92 2681578482
10 25613.91 64106186.77 2624363859
11 25613.91 61985757.24 2537558218
12 25613.91 58804911.06 2407341492
13 25613.91 55511967.40 2272535746
14 25613.91 51868335.18 2123373595
15 25613.91 49183678.11 2013469741
This tells me that the average RawIntDen in sample A is 2,96 times
higher than the average RawIntDen of sample B ([RawIntDen of sample A
from slide 1 to 15 /
RawIntDen of sample B from slide 1 to 15] / 15 slides). Is this correct
or nonsense? What
do you think?
Any help on this issue would be greatly appreciated.
Thank you very much!
Jakob
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