Nuclei Outline from DAPI pictures

[biscmm22]

Hi there,

I am a student working with HeLa cells.  I am using ImageJ to measure the difference in fluorescence in cell nuclei between different treatment groups.  My Dapi pictures are clear, and the nuclei are visible.  The location of the nucleus is less clear in the other pictures of protein (Ran, Topoisomerase, etc).  I am trying to find a way to outline the nuclei on the dapi pictures and then apply those outlines to the protein pictures.  I have tried multiple ways, but I can't seem to get it to work.  Is there a way that I could do this?

Thanks in advance for your help!

Sincerely,
Melissa

[FrankyG]

In all honesty your PI should be helping you with this and there are tutorials out there but hopefully this helps enough.

1) Open ImageJ

2) Open your desired image by dragging it over the ImageJ toolbar and dropping it

For the "Blue" Channel ONLY!
3) Click Image -> Color -> Split channels

4) Click Image -> Adjust -> Threshold

5) Check "Dark Background" checkbox

6) Use the first slider bar to make sure that the cells nuclei are filled with red

7) Click "Apply"

8) Click Process -> Binary -> Watershed

9) Click Analyze -> Analyze Particles

10) Use the following settings


11) Click "Ok"

12) Analyze your nuclei, make sure there are no micro-nuclei or overlapping ones, or ones that encircle multiple nuclei. If there are click on them in the image and then click "delete" in the ROImanager.

For the desired channel with the "protein" in it only!
13) You can then click on the image that has your proteins and click "Show all" in the ROImanager twice (once to click "off", another time to click "on" while you have the protein window selected!) and the overlay should apply to it.