errors in exported excel file using spot detcetor

[clara velazquez]

Hello!

I'm using the spot detector tool to measure the number of dots inside cells and I'm finding mismatches between the detection and the results exported to the excel file (pictures attached). Some times these numbers are quite different from the display in the picture and the excel but the most worry part is that sometimes in the excel file I have values >0 when there is no signal in that especific cell that at the end make me have colabeling of markers that cannot be expressed in the same cell.

Has some one observed the same problem? And do some one know why and how to fix it?

Thank you very much!

Clara

[Stephane]

Hi Clara,

Can you give us some details about your analysis workflow (it looks like you're using HK-Means plugin) so we can try to understand what happen ? We never observed that issue before so i guess sometime wrong happen at a specific step of the workflow. Thanks.

Best,

- Stephane

[clara velazquez]

Hi Stephane, 

Thank you for your answer. Here I send you more detailed screen shots. I am actually running an HK-means to distinguish the nuclei and then running the spot detector using ROIfixedfromSequence in the region of interest tab to limit the counting to that ROIs although some dots are also detected outside, not sure why.

Hope this can help you to understand why the excel file do not match with the display in the pictures.

Thanks!

Clara

[Stephane]

Hi Clara,

You need to use ROIFromSequence option, i tested it several time here to be sure and it works as expected :

The ROIFixedFromSequence is a very specific option you shouldn't use here.

If you still experience the problem we may need a sample image so we can reproduce it, thanks.

- Stephane

[clara velazquez]

Hi Stephane, 

Thank you very much for your help.

I've tried the ROIfromSequence option instead and now I cannot detect any signal. I've use the same sensitivity settings I was using yesterday and also modified them but have not been able to detect anything.

I'm attaching a screen shot and a couple of pictures  of the ones I have to analyze as an example. 

And one question... how can I limit the detection to the previous ROIs detected by using the HK-means? I had many spots detected outside the ROIs..

Thank you very much!

Clara

[Stephane]

Hi Clara,

Sorry for the late replay, I think i understand your problem now ! HK-Means automatically set the channel position of the ROI.

Before running the spot detector just change the channel position of your Cell ROIs to 'ALL', for that select all the ROIs (Ctrl+A) then set C position to -1 (= ALL) as in the picture :

Then spot detector will be able to find your spots from your cells. Hope that helps !

Best,

- Stephane

Le lundi 8 avril 2019 13:50:55 UTC+2, clara velazquez a écrit :