Posted by
Mark Hiner-2 on
URL: http://imagej.273.s1.nabble.com/Measuring-Fluorescence-density-of-nuclei-tp5008116p5008125.html
Hi Anoek,
This sounds fine, but how do I know what setting I should use?
>
If you run "Image > Adjust > Auto Threshold" and use the "Try all" method,
you will get a montage that displays the results of all the threshold
methods. You can use this result on a few sample images to guide your
selection. I suggest reading the wiki page for more information on the
individual threshold methods:
http://fiji.sc/Auto_ThresholdAnother question, if I'm using the threshold method, how can I do this the
> easiest/fastest?
>
You could certainly write a macro that thresholds and measures all of your
images. Take a look at the tutorial for macro programming:
http://fiji.sc/Introduction_into_Macro_ProgrammingBasically what you'll want to do is:
1) use the "Try all" auto thresholding to choose a threshold method
2) use the macro recorder (Plugins > Macros > Record...) to record the
analysis steps for a single image
3) adapt the Process_Folder template to apply your macro to all the images
in a directory.
You can access the Process_Folder template when you have a macro open in
the editor (e.g. via Plugins > New > Macro) by using the menu entry:
Templates > IJ1 Macro > Process Folder
And when I know the settings, should I just measure the whole picture?
>
The goal of thresholding here is to create a region of measurement
corresponding to the nuclei, so you wouldn't be measuring your complete
image.
And after this, is the Integraded Density my outcome or should I use a
> calculation?
>
I presume that you just want to measure the nuclei regions established via
thresholding. You can adjust what exactly is being measured via the Analyze
> Set Measurements option. You can read more about what each measurement
option represents here:
http://rsbweb.nih.gov/ij/docs/guide/146-30.html#sub:Set-Measurements...
Hope that helps!
- Mark
On Tue, Jun 10, 2014 at 1:04 PM, Anoekvanleeuwen <
[hidden email]> wrote:
> Hi,
>
> I have a question about measuring the amount of fluorescence with ImageJ.
> I stained my nuclei with DAPI and I'm trying to measure the fluorescence
> density, and with this the amount of DNA.
>
> First, I used a method to calculate the CTCF, where I drew a circle around
> the nucleus, and with CTRL + M I measured the area, mean grey value and
> Integrated Density. Unfortunately I found out that the size of the circle
> had influence on the CTCF, which in my opinion is not right.
>
> Then I heard of a 'threshold' method. I tried to find more about this
> method, but the only thing I can find is that I should use the option Image
> > Adjust > Threshold. This sounds fine, but how do I know what setting I
> should use? And when I know the settings, should I just measure the whole
> picture? And after this, is the Integraded Density my outcome or should I
> use a calculation?
>
> Another question, if I'm using the threshold method, how can I do this the
> easiest/fastest?
> I have around 1500 pictures each containing one or two nuclei.
>
> Thanks in advance!
> Anoek
>
>
>
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