Can anyone recommend me a method or software to quantify the average amount of PI staining in tumorsphere relative to the size of the tumorsphere by using confocal images?
Hi,
I assume that you have taken stacked confocal images of the 3D tumorsphere and want to count the number of PI stained cells?
It can be done via ImageJ. These are some methods mentioned in papers. This is one that use ImageJ to quantify viability through staining and confocal images for 3D culture. They also have a macro for that: Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M., Sprecher, C. M., & Ito, K. (2008). Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds. Tissue Engineering Part C: Methods, 14(4), 353-358.
Or you may search along with those keywords if you find the method makes sense but it is not ideal for you.
Alternatively, if IMARIS is available to you, it can be used to analyze 3D images directly. Basically you need to set an appropriate intensity threshold and generate particle volumes. Similar to ImageJ, there will be a whole list of particles generated (but it is 3D here), with information of sizes, intensities etc. Then just record the number of particles (that is the number of PI+ cells). Often, you will find 2 or more particles stuck together, then you may use different methods such as watershed to separate them. Overall, it is similar to 2D image analysis, and if you use a macro or program to count them, there will be some errors, but if you set the parameters/thresholds right and use them consistently, you should be able to get a fairly close number.
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