FIJI colocalization

Hey!

I have got some images (some channels are overexposed) to determine colocalization. Green channel shows the distribution of hormone (mostly cytoplasmic). DAPI and Actin staining is there. None are colocalizing perfectly. So what I need to measure is how many cells (DAPI positive) are positive for green and the intensity of green signal. Is this possible because of overexposure?

Any help/guidance is appreciated.

GunShan

Project_P0.5 HET 10X X5 1.tif (2.9 MB)
Project_P0.5 WT 10X 9X.tif (10.0 MB)

@Kushanlak

A few things now looking at your images…

A) Do you really want to do true colocalization analysis? From your description of what you want to do - I’d guess ‘no’. A basic workflow that I could envision here would be to:

  1. Segment your nuclei using the DAPI marker - results in a binary mask. (You can follow our Segmentation workshop and corresponding slides - these are the more updated slides.)
  2. Use the mask to measure the ‘green’ signal within… so intensity measurement within those regions. **NOTE - this would only measure the ‘green’ intensity within the nuclei areas… so as long as that is ok for you - that would be a good option.

(The workshop I linked there goes through exactly how to do this…)

B) Having the ‘green’ signal that you want to measure over-exposed is not good. Basically - you have lost information here by saturating the signal. You can use the HiLo LUT to see where your signal is maxed out and where it is ‘0’ (also not great - you are clipping on both ends).

C) Also - your WT versus HET - those images are two different sizes. Is there a reason you did not acquire them at the same resolution/size?

If I were you - I would reacquire these images. That is the only way you can reliably measure these intensity levels. Also - you need to take great care in your sample preps… do them all the same day using the same methods/reagents… acquire all samples on the same imaging system with the same exact settings. You can adjust things before you start to really acquire - being sure you don’t have over/under-exposure issues. Then go ahead and acquire all images at the same time - WT and experiment groups - and do not change any settings on the scope.

Maybe the Principles page is also helpful to read…

Hope this is clear. Let us know if you have more questions, etc.