Posted by
Swayne, Theresa C. on
URL: http://imagej.273.s1.nabble.com/macro-threshold-values-tp5015759p5015818.html
Hi Natalie,
It’s absolutely right to try to set a threshold in an unbiased way. It’s faster (in the long run) and more reproducible. The only difference folks are having is in how to find the most accurate method to select the threshold for this experiment.
You could use the same number for every image, or you could use a histogram-based method as you proposed, or you could use one of the automatic thresholding or trainable segmentation methods available in ImageJ.
The best approach will depend on your particular data — e.g. difference between signal and background, variability of the positive staining level, and variability in positive cell numbers, from one image to the next.
In the end, you will need to test your method empirically on several representative images from different groups.
--Theresa
On Mar 2, 2016, at 2:15 PM, Natalie M <
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[hidden email]>> wrote:
Thank you all for your feedback! The reason I am thresholding the values to
count the cells is because the cells I want to count have been stained with
fluorescent dye, and I want to count only cells with the brightest
fluorescence (i.e., only the cells which are positive for the actin I am
measuring). Instead of counting them by hand, I thought setting a global
threshold would eliminate any bias on my part and allow me to choose which
cells are the most brightly stained in each image. Does this still seem
like an accurate way of achieving the cell count, or would something
different be recommended?
-Natalie
On Wed, Mar 2, 2016 at 9:53 AM, Ewing James <
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[hidden email]>>
wrote:
------------------------------------
Theresa Swayne, Ph.D.
Manager
Confocal and Specialized Microscopy Shared Resource<
http://hiccc.columbia.edu/research/sharedresources/confocal>
Herbert Irving Comprehensive Cancer Center
Columbia University Medical Center
1130 St. Nicholas Ave., Room 222A
New York, NY 10032
Phone: 212-851-4613
[hidden email]<mailto:
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