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Hi everybody!
I have several doubts that I hope someone could help me with them:
We have a stack of images of cells stained with Red and Green dyes for TUNEL, and taken with confocal microscopy.
We want to calculate the proportion of red and green in each photo, in order to compare this data between groups of cells.
What I did was this: Each photo was changed to RGB channels. Then, in Color Threshold, I separated the R and G channels, choose a threshold and selected the particles. There are several threshold methods. I decided to use IJ_IsoData but in several photos (especially those red darker ones) I see that the Yen method is better (in terms of selecting better the particles). First doubt: May I change the method (and that changes the threshold number) depending on each photo, or choose the method and keep going without looking the threshold number, or manually set this number without choosing any method, until each particle (cell actually) is highlighted? Will that affect the results and later the statistical calculations?
About the red dark photos, or less bright... Should I make them brighter before the thresholding?
When analyzing particles, the table with total area, average size and mean data, are they in um^2 or in nm^2?
Is there any other way to count particles in red or green which you can recommend me?
Thanks a lot!
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