Posted by
Sindy Kueh on
URL: http://imagej.273.s1.nabble.com/Re-Counting-particle-fluorescence-labelled-using-ImageJ-tp3688447p3688451.html
Hi Damon,
I had a look at the macro.. Thanks. I haven't setup a new macro before
and am at a lost on where to begin... This may sound absolutely silly,
but do I input the whole macro as one, or as different macros....
Hope to hear from you soon..
Sindy
-----Original Message-----
From: ImageJ Interest Group [mailto:
[hidden email]] On Behalf Of
Damon Poburko
Sent: Thursday, April 29, 2010 6:42 AM
To:
[hidden email]
Subject: Re: Counting particle (fluorescence labelled) using ImageJ
Hi Sindy,
Here is the link to the macro.
http://rsb.info.nih.gov/ij/macros/Mulitple_Thresholds.txt It will
probably take a bit of playing with to get the parameters appropriately
set for your images. Have a gov with it, and it you're having problems,
feel free to drop me a line.
Cheers,
Damon
On 4/27/2010 11:39 PM, Sindy Kueh wrote:
> 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
> Process>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
>
>