Issues with mask/histoCAT segmentation

[cjauset]

Hello!

I've processed an image through the IMC Segmentation pipeline and I am now trying to analyze the single-cell data in histoCAT.
However, histoCAT only seem to recognize 5 cells in total, so I am not sure if my mask is properly created - it has a gradient of four intensities:

Mask

Screenshot 2020-08-12 at 17.56.54.png (484.81 KiB) Viewed 717 times

When I try to visualize channels in histoCAT using the heatmap I get the same gradient I see in the mask:

heatmap

Screenshot 2020-08-24 at 14.16.47.png (260.41 KiB) Viewed 717 times

If I do the step 3 of the segmentation pipeline, however, I get 126k cells. Is there any pipeline/script for downstream analyses using the single-cell measurements from the segmentation pipeline in histoCAT or R?

Thanks!

Best,
Cristina

[DenisSchapiro]

Dear Cristina,

I believe that the issue is that you are saving your mask as 16bit and you should create a 32bit mask instead.

If you have more than 65536 single cells a 16bit mask can not provide unique values to each cell. Just save it as a 32bit mask - histoCAT is supporting 32bit masks.

Best

Denis

[cjauset]

Ah, of course! Thanks Denis!

[cjauset]

Hi Denis,
I realized CellProfiler can just save 32bit images as floating points, but Histocat can only read masks in int32 or uint32 if I understood correctly. Is there a way to make this compatible?

Thanks again!
Cristina

[DenisSchapiro]

It should be able to load the CellProfiler 32bit float mask - or did you try and it fail?

[cjauset]

So after the transformation window when loading the images, nothing happens. There's no error message, but it doesn't say that it's loading either, so I assumed it was the mask format.