Yeast Cell & nuclei id, DAPI, mCherry, GFP

Image Analysis
,
#1

I am trying to construct a pipeline to compare staining of yeast cells.
The caveat is I am not a biologist, so
t3ImageTest.cppipe (22.3 KB)
his is the experiment as I understand it.

I have yeast cells grown under 2 stress conditions (salt/no salt).

I have been given 4 images all in color that I convert to grayscale:

a white field image
a blue DAPI stained image as a control for nuclear staining.
a red image which is a transcription factor tagged with mCherry
a green image with a transcription factor tagged with GFP

I need to figure out:

  1. What fraction of cells have a nuclear signal?
  2. What are the distribution of nuclear fluorescence values for stained nuclei?
  3. When either of the transcription factors are in the nucleus, what fraction
    of the time are both transcription factors in the nucleus?

I have attached the images and my pipeline.

I see on the forum that one is supposed to ID the nuclei first,
I have managed to do that, but I am having issues with getting
good cell identification, there are more cells divisions than expected.

How can I go from identifying nuclei to getting good cell id?
I switched to manual for the the first nuclei because I was unable
to get the auto methods to work.
I was unable to attach my images as they are too large.

Yeast nuclei colocalization
#2

I have the images available here:

https://uwmadison.box.com/s/jpe1s7tdch79yvfhw85tgcg4mrs6p097

#3

I found another pipeline on the forum that more or less does what I want.
I am having difficulty getting the 2nd MaskObjects to mark regions, it seems to ignore
what look to be reasonable overlap? Any suggestions would be appreciated. Thanks

MyTest-Pipeline.cppipe (31.3 KB)

#4

Since I am starting with 3 separate images, do I need to align images in this pipeline?

#5

Note this post is a repeat of this one: Yeast nuclei colocalization