need help thresholding background for particle analysis

[Stefaniak, Aleksandr (CDC/NIOSH/DRDS)]

I am trying to use Image J to measure the size of particles that were deposited onto a polycarbonate membrane, image 'Ag-L-1.tif'.  When I try to threshold the image (auto or manually), the software treats the particles and the visible pores in the membrane the same so I am left with black spots of the particles and black halos from the membrane pores 'Ag-L-1 (thresholded.tiff)'.  When I try to size, the software measures the halos of the membrane pores as if they were particles and also measures any halos near the black spots of the particles as if they were all the particle.

Any guidance on how to threshold out the background membrane pores to leave only the particles for measuring would be extremely helpful.

Thank you,

Aleks



Aleksandr Stefaniak, PhD, CIH
Division of Respiratory Disease Studies
National Institute for Occupational Safety and Health
Division of Respiratory Disease Studies
1095 Willowdale Road
Morgantown, WV 26505
Tel: [phone]
Fax: [phone]

[Rob van 't Hof]

hi Aleks,
Your image is a bit low resolution for accurate measurements. However,
this little macro seems to do the job reasonably well:

selectWindow("Ag-L-1b.tif");
run("Duplicate...", "title=Ag-L-1.tif");
run("8-bit");
//run("Threshold...");
setAutoThreshold("Default dark");
setThreshold(145, 255);
run("Convert to Mask");
run("Options...", "iterations=1 count=1 edm=Overwrite do=Nothing");
run("Close-");
run("Options...", "iterations=2 count=1 edm=Overwrite do=Nothing");
run("Open");
run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00
show=Nothing display clear add");
selectWindow("Ag-L-1b.tif");
run("From ROI Manager");

Hope this helps,
Rob


On 29/06/2011 15:02, Stefaniak, Aleksandr (CDC/NIOSH/DRDS) wrote:

> I am trying to use Image J to measure the size of particles that were deposited onto a polycarbonate membrane, image 'Ag-L-1.tif'.  When I try to threshold the image (auto or manually), the software treats the particles and the visible pores in the membrane the same so I am left with black spots of the particles and black halos from the membrane pores 'Ag-L-1 (thresholded.tiff)'.  When I try to size, the software measures the halos of the membrane pores as if they were particles and also measures any halos near the black spots of the particles as if they were all the particle.
>
> Any guidance on how to threshold out the background membrane pores to leave only the particles for measuring would be extremely helpful.
>
> Thank you,
>
> Aleks
>
>
>
> Aleksandr Stefaniak, PhD, CIH
> Division of Respiratory Disease Studies
> National Institute for Occupational Safety and Health
> Division of Respiratory Disease Studies
> 1095 Willowdale Road
> Morgantown, WV 26505
> Tel: [phone]
> Fax: [phone]
>
>
>

--
_____________________________
Dr. Rob van 't Hof
Senior Lecturer
Bone Group
Molecular Medicine Centre
Western General Hospital
Edinburgh EH4 2XU
UK

Phone: [phone]
email: [hidden email]
_____________________________



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