I'm looking for recommendations or citations for good computational methods for thresholding and counting cell bodies in fixed tissue samples based on a nuclear stain such as DAPI. Simple thresholding and connected components is somewhat problematic because these stains tend to be somewhat fragmented. Perhaps thresholding followed by a dilation/erosion cycle would do the trick... Any suggestions?
No. If you do not find anything on pubmed or google I would suggest, that the test you look for does not exist. In my point of view it sounds a little too complicated. Especially thinking about focus and positive/negative control including the standard dilutions.
try this article may it will help
http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=115241
Fragmented signal is an important issue specially if going for automation. Uneven staining may lead to inadequate and disproportionate thresholding, that in turn may cause fragmented selections which when counted may result in erroneous data. One way to go around this problem is to increase DAPI concentration. Increase the DAPI concentration as long as it does not bind to non target structures and optimize it to a level that all nuclei show similar HSB values. (The blue nuclei will be lot more blue or uniformly dark blue, this is ok for computer as long as stain go off target structures.) Subsequently, you can as well also raise or decrease the threshold value until it matches with your visual preferences. Idea here is to get the computer accept all the borderline Hues of the stain , so that it reduces the fragmented selections while thresholding. For automation, try macros in Image J or actions in photoshop, both of them save a lot of time.
What I ve done with IHC tissue samples was to use a plugin in ImageJ where I could mark nuclei with green or red dots and get them counted by the software. I know it sounds a bit objective or time consuming but his is the best solution to my knowledge.
http://www.youtube.com/watch?v=PqHFsmS1_JY
I don't know if this can help you.
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