http://imagej.273.s1.nabble.com/Measuring-Fluorescence-density-of-nuclei-tp5008116p5008880.html
The reason we were talking about fluorescence came from the title of the email... If this is a different question, it would be advisable to change it.
I am still unsure on what you mean by optical imaging and activity maps. Is the activity map an image or some computer rendition of another acquisition tool, like EEG data? The best approach would be for you to provide an example image....
Otherwise, a remark: If the areas with higher density are dark, then it would make sense that the mean value is small, no? Dark: low values and bright:high values.
If you want the values to be inverted then just use "Edit"->"Invert", then your average intensities will be higher when there is a higher density.
> -----Original Message-----
> From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of
> nmichae
> Sent: jeudi 24 juillet 2014 11:48
> To:
[hidden email]
> Subject: Re: Measuring Fluorescence density of nuclei
>
> Hello Oliver,
>
> Thank you for the message. But what Im doing is not fluroescence measurement
> but we do optical imaging. and we get the neuronal activity in a grey scale
> activity map. So my intention was to measure the density of this activity patch.
> So where i expect a high density or where I have a darker activity patch, which
> denotes high brain activity, I 'm getting a small measurement value when I use
> this tool in imageJ. Thats why I was wondering, what actually is being measured
> here. Im very bad in explaining.
> Dont know whether you will understand what Im trying to say.
>
> Regards,
>
> Neethu
>
>
> On Thu, Jul 24, 2014 at 10:28 AM, Olivier Burri [via ImageJ] <
>
[hidden email]> wrote:
>
> > Hi Neethu and all,
> >
> > First a couple of questions:
> > - What kind of microscope are you using? CONFOCALS are usually a
> > no-go for 'fluorescence intensity' measurements. If all the cells are
> > acquired at the same distance from the coverslip and the same volume
> > is taken, you might have a chance, but I have not yet seen data confirming
> this.
> > - Have you kept all imaging and sample preparation parameters
> > identical over each of your 1500 images?
> > - How were your stainings done?
> >
> > As a good starter, I'd suggest you have a quick read at:
> >
> > *Seeing is believing? A beginners' guide to practical pitfalls in
> > image
> > acquisition*
> >
http://jcb.rupress.org/content/172/1/9.full> > The following paragraph should give you some information:
> > *Quantification of images—why is it useful and when is it
> > appropriate?*
> >
> > Fluorescence signal is very linear in WIDEFIELD microscopy and
> > correlates well with fluorophore concentration, up to a point which
> > makes it OK for semi-quantitative or relative measurements. If you
> > expect a 5 fold or more difference then it should be OK.
> >
> > Also remember that DAPI only stains AT and not GC, so depending on
> > your biological question, this could affect any interpretation you
> > would like to draw from your experiments. And with a quick search I
> > have not found any papers claiming that light microscopy yields
> > information on DNA concentration from DAPI stainings. The closest I
> > have found, and am likely to believe, is using FACS.
> > *Critical Aspects in Analysis of Cellular DNA Content*
> >
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976661/> >
> > Also, when working on thresholds to measure intensities, it’s good
> > practice to use a 'neutral' stain to detect the area. Setting a
> > threshold on the intensity to then measure that same intensity biases the
> results.
> > One stain marks the area independently of the stain from which you
> > would like to extract the intensity information
> >
> > In the end, fluorescence microscopy like the one you are performing is
> > good for answering "Where is this?", not so much for "How much of this
> > is there?"
> >
> > Finally, if you still want to go about with this, an idea would be
> > something in the lines of:
> >
> > 1. Blur your image significantly to smooth out uneven DAPI patches.
> >
> > 2. Apply a "Find maxima" filter to get a single point per nucleus.
> >
> > 3. Enlarge these points to get a surface of a fixed size that fits
> > inside the smallest nucleus you can find.
> >
> > Measure these surfaces (No threshold) on your non-blurred image (You
> > can also measure it in your blurred image). Integrated density and
> > average density should yield the same when you divide the integrated
> > density by the area.
> >
> > Make sure you have controls and/or at least two conditions to compare!
> > Just absolute differences will mean nothing unless you have something
> > to compare them to. Something like fibroblasts vs. tumor cells where
> > you KNOW you will get a difference. You can thus also assess the
> > natural variation to expect from a given sample.
> >
> > This was by no means a message to discourage you, but just to inform
> > you of the potential dangers and critiques that using DAPI
> > quantification could bring.
> >
> > All the best!
> >
> > Oli
> >
> > Olivier Burri
> > Engineer - Image Processing
> > & Software Development
> > EPFL - SV - PTECH - PTBIOP
> >
> > --
> > ImageJ mailing list:
http://imagej.nih.gov/ij/list.html> >
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