http://imagej.273.s1.nabble.com/Re-Counting-particle-fluorescence-labelled-using-ImageJ-tp3688447p3688450.html
Here is the link to the macro.
set for your images. Have a gov with it, and it you're having problems,
> Hi Damon,
>
> Would like to thanks you again for the clear instruction. I know it has
> been a while ago, but I finally tried performing the analysis as you had
> suggested. I am still learning how to use ImageJ and indeed it is a very
> useful software if used to its capacity.
> Anyway, yes, I am interested in the macro you mentioned in your earlier
> email to capture individual neighbouring puncta which might otherwise
> become a blob using a single treshold.
>
> With best regards
> Sindy
>
>
> Sindy Kueh
> Anatomy and Cell Biology
> Room 30.2.09 University of Western Sydney
> School of Medicine
> Building 30 Goldsmith Ave
> Campbelltown Campus NSW 2560
> Locked Bag 1797 Penrith South DC NSW 1797
> Tel (02) 9852 4722
> Fax (02) 9852 4701
>
> -----Original Message-----
> From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of
> Damon Poburko
> Sent: Wednesday, February 03, 2010 4:26 AM
> To:
[hidden email]
> Subject: Re: Counting particle (fluorescence labelled) using ImageJ
>
> Hi Sindy,
>
> It sounds like you have probably found the Analyze>Analyze Particles
> function, which is a good place to start. Probably the most important
> aspect that you will have to set, other than the threshold, will be your
> minimum and maximum particle size. If you images are scaled (i.e. in
> microns, etc), the units will be square microns, otherwise it will just
> be in pixels (which is more intuitive). You can get an idea of the sizes
> that make sense for you particles by tracing a few by hand and measuring
> their area. (i.e. trace, add to the ROI manager ("T" is the shortcut),
> repeat for other clusters, then set your measurements to include area).
>
> As for the thresholding, there are a few things to consider.
> 1. Running a Gaussian filter (Process>Filters>Gaussian Blur...) will
> help remove noise. This is a handy start to almost any process involving
> thresholding. (I applied a Blur of Sigma=0.9 to your image and counted
> ~600 particles with a modest threshold) 2. Is you background uniform? If
> not, you could consider using
> Process>Subtract Background.. with a ball size a little large than your
> clusters. This effectively tries to locally subtract the background
> while preserving bright puncta smaller than the size of the rolling
> ball.
> 3. Finally, you could consider a method that applied multiple thresholds
> to your image and counts clusters/puncta that are restricted to certain
> size or shape criteria. This becomes quite usefull in the case of
> closely neighbouring puncta that will sometimes turn into one big glob
> when using a single threshold that is low enough to let you capture
> somewhat dim puncta. If you are interested I will send you just such a
> macro.
>
> Best of luck,
> Damon
>
>