# Two channels, 1 set of objects separated by intensity

Hello and thank you in advance!

I have two channels that contain the same object. I wanted to divide each individual cell to latter obtain the mean for these objects using calcmath-measured by mean intensity. The problem is, I do not know if Calculate Math is doing this.

So over one object there are many intensities. I want to make sure that calcmath is taking each object intensity of channel 1 then dividing it by the same object intensity of channel 2 then finding the mean of the over the entire object, rather than taking the object intensity of channel 1, finding the mean, then dividing object intensity of channel 2’s mean.

This may sound a bit confusing, haha.

Hi,

If I understand what you’re saying correctly then what you want is the following:

• Use ImageMath to divide the channel1 image by the channel2 image to yield a new image. Make sure to disable capping the values at 0 and 1.
• Use MeasureObjectIntensity to measure the intensities of your objects against the output of ImageMath. The mean per-object intensity will be one of several measurements collected.

This should perform the operation you want. However, dividing the per-object mean intensity of channel1 by the per-object mean intensity of channel2 should be mathematically equivalent, so you can also do this:

• MeasureObjectIntensity of the objects from channel1 and channel2
• CalculateMath to divide the mean per-object intensity from channel1 by that of channel2

The important thing in each approach is to measure from the same objects for both channels. Do you have reason to believe these are not the same?

Regards,
-Mark

Yes! Exactly. I have started to do that. The only problem I am running into is that when I look at the cell math compared to what I manually figured, the ratios are not aligning, and I am quite curious to know what exactly the Calculate math is finding.

Edit: Do I just realized what math it was taking.