Help with intensity

Hi,

I’ve been trying to figure out how to obtain the average intensity of the channel 2 using the area identified by channel 1. I am sort of a beginner and have searched the forums for help but haven’t figured it out completely.

I used automatic thresholding and a size cutoff of 10 to 200 but there are some faint dots that it is not picking up in channel 1. So I haven’t gotten past this stage. I then would assume that I have to use the outliens for this image to get the intensities from the second channel? If so, what is the best way? Mask? Thanks!

For some reason, it is not letting me upload my files. Are they too big? They are 1.6 MB TIff images

dropbox.com/s/e29gabt0e0yyo … 1.tif?dl=0

dropbox.com/s/oqmramams8sxf … 2.tif?dl=0

For this purpose, you would use the MeasureObjectIntensity module. You create an object set, typically using IdentifyPrimaryObjects or IdentifySecondaryObjects, and use that object as one of the inputs into MeasureObjectIntensity. The other input is the image (i.e., channel) you want to measure from. You can measure from any combination of objects and channels, and from multiple objects and multiple channels. The mean intensity of the pixels within each object is one of the outputs of this module, among others.

Regards,
-Mark

Thanks. And how do you measure the intensity of the part of channel 2 NOT outlined by channel 1? I’m trying to mask it but not really working.

Figured it out ;D

Found out how to mask the image but I’m having issues identifying those objects. When I identify primary objects for this new masked image, it identifies some, but misses a lot of the lower-intensity cells. It’s a tissue section so maybe it can’t recognize discrete cells? Is there a way to get around this? Why can’t i just have the program measure the intensity of the masked image? Do I have to identify objects first?

Wow…figured out that too. Sorry for all the spam.

So the output for the mean intensity of the image, is it including the black pixels as well? In that case, I definitely need to identify objects then so that the black pixels are not included :frowning: So I guess this goes back to my question 2 before this one. Thanks!

I think I may be confused by what you have/have not done by this stage. I have the two images you uploaded, but can you also upload the pipeline you have thus far, and describe precisely what regions you want the intensity from?
-Mark