Posted by
Olivier Burri on
Sep 13, 2012; 8:12am
URL: http://imagej.273.s1.nabble.com/Fluorescence-intensity-analysis-with-tissues-tp5000037p5000052.html
Dear Sebastian,
> The quantative analysis of immunostained specimen troubles me and unfortunately the few things which I could find on google and in this forum did not match my concerns, so I deceided to open a new thread and ask for help.
First off thank you for the post and the details regarding your
experiment. Very often we see people using fluorescence quantification
without thinking too much about whether they are allowed to do it or
not.
> I started by taking pictures of the islets on my slides and used exactly the same settings (time, gain, gamma, ...). The same was done with blank slides (without primary antibody).
>
> In Image J I used the image calculator to substract the picture from the blank slide from the picture I want to analyse in order to remove background.
>
> Then I selected the islet area with the freehand tool and measured the mean grey value.
> 1) Can you do it like this or did I miss something?
From what you say in 3) you seem to have a very large difference
between your two conditions, so this should be doable. Some
considerations though.
1) How many different replicates do you have? (Of the same animal, of
other animals prepared in another batch). Ideally I think it would be
good if you assessed the variability within the same animal and the
variability between animals for both your WT and "treated" conditions.
That way, hopefully the difference you find between the conditions
will be larger than the uncertainty and you can be safer in your
interpretations.
2) Did you perform your stainings in batch, so as to minimize
variabiliy coming from the staining protocol?
> 2) I read that people normalize the intensity with the area when working with cells. Should you also do that when working with tissue and if yes, how is the best way to do it?
Usually when you normalize with the area it's to get a density
measurement, and is part of a different type of experiment where you
find your cell area (threshold) and you normalize that with the tissue
area to estimate the density of your sample. I am not sure about
measuring intensities inside cells (Using a threshold I guess) and
then normalizing the intensities. You'd get something like Pixel
Intensities / um2 which doesn't mean much more than just average
intensity...
When you normalize intensity measurements, you do it as ratios of
signals coming from within your own sample, like when you check for
the presence of actin on Western blots. Do you have some marker you
could use as an internal control? Some cytoplasmic marker for example,
that should be homogeneous no matter the condition, that would allow
for normalizing your signal to this one. It can also be a good
indicator that the staining protocol happened correctly.
> 3) What about using a threshold in order to get clearer results? I thought about it, but I got pictures with very low staining and others which very high intensity, so I could not use the same threshold for all pictures. I wonder whether it's a good idea or even scientifically correct to set the threshold to the mean grey value +/- 50 % on every picture or something like that.
A way we try it out here is to test out all the available automatic
thresholds available on Fiji/ImageJ using a macro. First the user sets
the threshold manually on a set of images (about 10 or more). Then the
plugin launches the auto-threshold methods on the images and recovers
their values. Then the plugin compares your values to the ones that
were automatically selected and recommends a threshold and a
correction factor that could be used (Something that is done in Cell
Profiler, for example).
However thresholds are usually not set on the data you want to measure
but on some other channel that acts as a mask to your data. Something
that only stains the tissue or cells that you are interested in.
>
> Maybe someone can give me some inspiration, thank you in advance! :)
Hope that's a bit of inspiration. If you'd like the little macro I was
mentioning, let me know. I'd have to comment it a bit to make it
useable but would be happy to share it.
Best
Oli
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