Posted by
jchanson on
Aug 10, 2011; 2:09pm
URL: http://imagej.273.s1.nabble.com/Counting-spots-within-nuclei-tp3683546p3683547.html
David -
One more option for counting spots in nuclei is to use the find
maxima to find a single point for each protein spot and then use the
redirect option with analyze particles to measure the intensity from the
single point image. This "intensity" will correlate to the number of
protein spots.
Here's a macro for it. You'll need to add your own threshold or
segmentation for the DAPI image. And, you'll need to set the "noise"
tolerance on the protein image to whatever works for your images...
// Select images to analyze
waitForUser("Click on DAPI image");
dapiID = getImageID();
waitForUser("Click on protein image");
proteinID = getImageID();
// Reduce protein image to a single point for each spot
selectImage(proteinID);
run("Find Maxima...", "noise=10 output=[Single Points]");
rename("Protein Points");
selectWindow("Protein Points");
proteinpointID = getImageID();
// Instead of "255" as the value of each point, reduce them to "1" for
each point
selectImage(proteinpointID);
run("Divide...", "value=255");
// Use redirection with analyze particles and integrated density to count
number of spots per nuclei
selectImage(dapiID);
run("Set Measurements...", "area integrated redirect=[Protein Points]
decimal=3");
run("Analyze Particles...", "size=100-Infinity circularity=0.00-1.00
show=Nothing display");
// Close the "single protein points" image
selectImage(proteinpointID);
close();
Jeff Hanson
Senior Imaging Analyst
Eli Lilly and Company
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Subject
Counting spots within nuclei
I need some help with a project one of my users would like to do. They
are doing medium throughput screening for nuclear segregation defects. The
samples are stained and then a nuclear image (DAPI) and a protein
immunofluorescence image is collected. In wild-type there is one protein
spot per nucleus. In a mutant, there are multiple spots per nucleus or
none. THere are 50-100 nuclei per image and each image represents a
putative mutant. We have used the Find Maximum command to effectively
count the total number of spots per field, but it turns out to not be
enough information (since some with two spots and some with zero spots is
the same as wild-type). They would like to generate a histogram of the
distribution of spot numbers per nucleus for each image since a mutant
would be skewed away from one. Is there a way to use the nuclear image to
define the area of each nucleus and then count spots in each individual
nucleus so that the distribution can be plotted in Excel etc.? Thanks-
Dave
Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)