Re: Colocalization analysis w/ ICA and JACoP - No intensity saturation please
Posted by
sibert on
Jun 03, 2010; 3:19pm
URL: http://imagej.273.s1.nabble.com/Re-Colocalization-analysis-w-ICA-and-JACoP-No-intensity-saturation-please-tp3688067p3688069.html
Hello,
Daniel James White wrote
Looking at cell1_Green.tiff and the red one that goers with it,
it is clear that the images are very badly over exposed / clipped / saturated.
in cell2 the green is not saturated, but there is a saturated pixel in the red channel.
(its significant as the image is small)
cell3 is badly saturated.
This is very very bad news in general for quantitative analysis and especially for coloc analysis.
Yuo cant use images like this in this kinf of analysis.
You will get false/meaningless results.
Certainly these images could not be quantified, they were not taken with such intent. At this point they are the only ones I had on hand to begin testing methods of analysis. I wanted to make sure I could analyze the images properly before collecting them.
I did not think poor image quality would result in differences between quantification methods, but perhaps they handle saturated and empty pixels differently? This would be important to know.
I've found a few sample colocalization image sets online and I have begun using those to test various methods of analysis. It appears that there are still some differences between the ICA and JACoP plugins, although primarily in how they apply thresholds. I would like to understand better what is going on; I think this has important implications for interpreting the results. For example, Li et al. emphasize that 0-0 pixel pairs should not be counted in ICQ analysis. The ICA documentation states that the lower threshold must be >0 and the upper threshold must be set to 255, but I do not know how JACoP addresses this.
I appreciate the link, I have also found the following article to be very helpful in describing imaging requirements and various methods of quantification.
http://labs.pbrc.edu/cellbiology/documents/Colocalizationtutorial.pdf-Thanks,
Bryan