http://imagej.273.s1.nabble.com/Re-Counting-particle-fluorescence-labelled-using-ImageJ-tp3688447p3688452.html
Here's what you need to do. Copy the macro txt file into your
Image>Plugins>Macros folder in your file explorer. The next time you
open image J, you should see the macro in the Plugins>Macros sub-menu.
Simply click in the macro, and away you go. If that's not making sense,
> 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
>>
>>
>>