How to count cells with different colors in a single image?

[Antonio Nour]

  Hello, good people of Icy

 I have been trying to use Icy to count pink (Ki67) and blue (DAPI) cells in a series of images, but with no success. 

My goal is to determine the proportion between pink and blue cells on the images. I have already tried using the spot detector and using the channel selection to differentiate between those two colors, but the app doesn't seem to do it. Am I using the right tool? If so, am I using it right?

What I've been doing is: Open the image, open spot detector, set channel to different values (none of them work), set size filtering, and start detection. But random dots appear instead of the detection of cells I would like.

Any help is much appreciated.

[Stephane]

Hi Antonio,

Do you have the original image ? Here you're sharing a JPG file where pink color is shared between the blue and red channel, making separation of the 2 signals impossible :
- red channel give you Ki67
- blue channel give you Ki67+ DAPI

Having the original image would allow to separate both signals correctly and so allow allow your quantification.
About Spot Detector, i guess you didn't used the correct parameters, you need to correctly set the image type (fluorescence image) and scale level (see the attached picture).

Hope that helps.

Best,

- Stephane

[Antonio Nour]

Hello Stephane

Thank you so much for replying! It realy helped!

The screenshot you posted represents exactly what I need, as Icy identifies the pink cells in the image. I was probably not configuring the Spot Detector how it should, but it now works like a charm. I now need to quantify the blue cells in the images.

The JPG I posted is a merge between two other images, which I will be posting here. As you pointed out, it isn't possible to configure the SD to detect the blue cells, although I don't fully comprehend why.

Thank you again for the kindness of helping!

Antonio

[Stephane]

Hello Antonio,

Well in fact you can also do the detection in blue channel, just select correct channel in "Pre Processing" section.
For your JPG image ,you have to select "channel 2" (3rd channel) then use the same parameter as for the other channel except you may need to increase sensitivity to 200 (as you have low intensity spot there), see attached picture.

Also you can do XLS export directly through the Spo Detector (see options in output section), ot use ROI export then you can export the ROI statistics in excel directly from Icy :)

Hope that help.

Best,

- Stephane