http://imagej.273.s1.nabble.com/Problem-regarding-cell-counting-tp5007416p5007423.html
On Thu, Apr 24, 2014 at 1:26 PM, JOEL B. SHEFFIELD <
> Hi Sarah,
>
> Here's another approach. First, I separated the channels --no need to work
> with the red and blue channels, since they have no data anyway. Then, I
> ran the FFT bandpass filter to smooth out the background. The idea is that
> the variation in the background is some sort of low frequency wave
> function, and you can exclude it from the image. I then took the resulting
> image and ran a threshold on it, using Image>Adjust>Adjust threshold, and
> selected something that seemed to capture all of the cells. I then went to
> Edit>Selection>make mask to create a separate mask derived from the
> thresholded image. I then ran Process>binary>watershed to reduce the cell
> clusters to individuals. This was followed by Edit>selection>make
> selection (on the binary image). I then selected the original image, and
> clicked on Edit>selection>restore selection. This gave me the first image
> that you can see. Note that it includes most of the cells, but also lots
> of smaller objects. I then used Analyze Particles, having first selected
> Area in set measurements. In the Analyze Particles menu, I deselected
> "ignore edges" and "ignore holes". I also restricted the size to between
> 1000 and infinity. That elminates the small stuff. I also asked it to
> display outlines. You can see the result in the second image. I Counted
> 90 cells in the first run, and 77 in the second. The difference is due to
> how I set the thresholds. That can be tricky.
>
> Here is the macro sequence, but it needs fleshing out.
>
> run("Split Channels");
> selectWindow("447_1.4_40XRGB_LeftBA_-1.84Bregma_c2.JPG (blue)");
> close();
> selectWindow("447_1.4_40XRGB_LeftBA_-1.84Bregma_c2.JPG (green)");
> selectWindow("447_1.4_40XRGB_LeftBA_-1.84Bregma_c2.JPG (red)");
> close();
> selectWindow("447_1.4_40XRGB_LeftBA_-1.84Bregma_c2.JPG (green)");
> run("Bandpass Filter...", "filter_large=200 filter_small=0 suppress=None
> tolerance=5");
> setAutoThreshold("Default");
> //run("Threshold...");
> run("Create Mask");
> run("Watershed");
> run("Create Selection");
> selectWindow("447_1.4_40XRGB_LeftBA_-1.84Bregma_c2.JPG (green)");
> run("Restore Selection");
> run("Set Measurements...", "area mean redirect=None decimal=3");
> selectWindow("447_1.4_40XRGB_LeftBA_-1.84Bregma_c2.JPG (green)");
> run("Analyze Particles...", "size=200-Infinity show=Outlines display
> clear");
> close();
> run("Analyze Particles...", "size=1000-Infinity show=Outlines display
> clear");
>
> Joel
> [image: Inline image 3][image: Inline image 1]
>
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail:
[hidden email]
> URL:
http://astro.temple.edu/~jbs>
>
> On Thu, Apr 24, 2014 at 3:19 PM, SARAH ROSE STERLACE <
>
[hidden email]> wrote:
>
> > Thank you so much your reply! The macro works pretty well, but
> conservative
> > for the positive pictures, however, I also have neighboring regions (to
> > show the limited spread) that have no fluorescent neurons stained, but do
> > contain "dust." (or autofluorescence). It seems to recognize and
> > overestimate in these regions. I have attached a picture of the
> > neighboring regions. When I applied your macro, it came up with 38.
> > Any thoughts?
> > I am relatively new to Image J, so please let me know if this is because
> of
> > "user error."
> >
> > thanks!
> >
> >
> >
> > On Thu, Apr 24, 2014 at 11:15 AM, BioVoxxel <
[hidden email]
> > >wrote:
> >
> > > Hi Sarah,
> > >
> > > You might try the following short macro:
> > >
> > > run("Subtract Background...", "rolling=100");
> > > run("Median...", "radius=12");
> > > run("Minimum...", "radius=8");
> > > run("Find Maxima...", "noise=2 output=[Count] exclude");
> > > run("Find Maxima...", "noise=2 output=[Point Selection] exclude");
> > > run("Revert");
> > >
> > > If your images are all similar, this might be able to recognize mist of
> > > your cells and count them.
> > >
> > > regards,
> > > Jan
> > >
> > >
> > > 2014-04-24 18:54 GMT+02:00 SARAH ROSE STERLACE <
>
[hidden email]
> > >:
> > >
> > > > Hello,
> > > > I am having trouble how to figure out a way to automatically count
> the
> > > GFP
> > > > stain without counting the background. I have been using the ITCN
> > plugin,
> > > > but I cannot figure out how to have it just count the circles. Any
> > help
> > > > would be greatly appreciated.
> > > > I have been inverting the image, setting it to 8-bit and enhancing
> the
> > > > contrast by 3% to show the cells stained deeper in the tissue better
> to
> > > see
> > > > if that would help. (i have not attached that image because it
> exceeds
> > > the
> > > > size for the email) I am attaching an original image. I have about
> 100
> > > > images to count, so if I could figure out how to automate the
> counting
> > > that
> > > > would be awesome!
> > > >
> > > > --
> > > > Sarah R Sterlace, C. Phil
> > > >
> > > > --
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> >
> >
> >
> > --
> > Sarah R Sterlace, C. Phil
> > my email address:
[hidden email]
> >
> > Behavioral Neuroscience
> > UCLA
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Sarah R Sterlace, C. Phil