(no subject)

[Merlyn Emmanuel]

Dear Image J Users,

I am trying to count  beta1 vesicles (objects) in my images and want to
quantify the numbers of beta1 vesicles in mock and knock down cells.

I am facing the following problem,

Since integrin stains the intracellular vesicles as well as the plasma
membrane very strongly, the software is picking up the stained membrane as
objects and i do not wish to include them in my calculation. so is there
any way to exclude the plasma membrane staining while it is counting the
vesicles over the image?

Also is there any script to calculate the vesicle numbers over a set of
images like in batch mode?

Best,
Merlyn

--
Regards,

Merlyn Emmanuel
c/o Dr. Sunando Datta
Dept. of Biological Sciences
IISER Bhopal
INDIA

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[ctrueden]

Hi Merlyn,

> Since integrin stains the intracellular vesicles as well as the plasma
> membrane very strongly, the software is picking up the stained
> membrane as objects and i do not wish to include them in my
> calculation. so is there any way to exclude the plasma membrane
> staining while it is counting the vesicles over the image?

Check out this page:
http://imagej.net/Segmentation

And in particular, you could try the Trainable Weka Segmentation (TWS)
plugin:
http://imagej.net/Trainable_Weka_Segmentation

Regards,
Curtis

On Wed, May 27, 2015 at 12:26 AM, Merlyn Emmanuel <[hidden email]>
wrote:

> Dear Image J Users,
>
> I am trying to count  beta1 vesicles (objects) in my images and want to
> quantify the numbers of beta1 vesicles in mock and knock down cells.
>
> I am facing the following problem,
>
> Since integrin stains the intracellular vesicles as well as the plasma
> membrane very strongly, the software is picking up the stained membrane as
> objects and i do not wish to include them in my calculation. so is there
> any way to exclude the plasma membrane staining while it is counting the
> vesicles over the image?
>
> Also is there any script to calculate the vesicle numbers over a set of
> images like in batch mode?
>
> Best,
> Merlyn
>
> --
> Regards,
>
> Merlyn Emmanuel
> c/o Dr. Sunando Datta
> Dept. of Biological Sciences
> IISER Bhopal
> INDIA
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html
>

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[Greg]

Hi Merlyn,

as I am no expert in your specific biological problem, I can just point you to some hopefully plausible strategies.

So when both the membrane and the vesicles are stained, that means to me, that you will get comparable intensities for both structures, therefore thresholding alone wont help you. What you might use to distinguish them, is their shape. So for example you could, after thresholding, go to Analyze -> Analyze Particles.. and choose a circularity and/or a size range which excludes the membrane. A more advanced approach which goes in the same direction is to use some morphological transformations to filter out the undesired membrane features. Look for example at this plugin

http://rsb.info.nih.gov/ij/plugins/gray-morphology.html

And here is a brief but pointed introduction to morphological transformations for image analysis:

http://homepages.inf.ed.ac.uk/rbf/HIPR2/morops.htm

In my head I have of course some circular vesicles and some irregular membrane patches. You could also upload an example image if it is a completely different scenario.

Best,
Greg