Counting spots within nuclei

Posted by Knecht, David on Aug 06, 2011; 11:26am
URL: http://imagej.273.s1.nabble.com/Counting-spots-within-nuclei-tp3683546.html

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)