Posted by
Sindy Kueh on
Apr 28, 2010; 6:39am
URL: http://imagej.273.s1.nabble.com/Re-Counting-particle-fluorescence-labelled-using-ImageJ-tp3688447p3688449.html
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
--
Damon Poburko, PhD
Postdoctoral Research Fellow
Stanford University School of Medicine
Dept. of Molecular & Cellular Physiology
279 Campus Dr., Beckman B103, Stanford, CA 94305
Ph: 650 725 7564, fax: 650 725 8021
Sindy Kueh wrote:
> Hi all
>
> I am a newbie with using imageJ and am trying to count the number of
> clusters and also to measure the cluster size of GABAA receptor a1
> subunit (fluorescence labelled). I have tried looking up on the ImageJ
> homepage and could not find or do not know where to look for
> information on how to correctly set threshold etc on counting these
> clusters/particles. I have downloaded a 2 page pdf file which explains
> how to count particle etc, but have difficulty locating the
> file-embryos in which is needed to do the "tutorial".
> I read that in order to perform the analysis, I have to change the
> file type to either 16-bit or 8 bit-gray scale, which I did...
> however, I had difficulty trying to set the threshold and have
> returned results in the order of thousands of clusters where only a
> few hundreds are present. I have attached an example of a file of what
> I need to measure... the clusters are labelled green.
> I hope someone can help.
>
> Many thanks
>
> Best regards
> Sindy
>
>
>
> <<Example1.JPG>>
>
>
>