are red traces from two images comparable?

[XiaoqianSun0104]

Hi,

I have two recordings from two channels (Ch1 and Ch2) of same ROI:
- Neurons in Ch1 and Ch2 have certain level of overlap.
- Neurons in Ch1 have normal activity, while neurons in Ch2 should have a stable but weak signal

After using CaImAn to process Ch1/Ch2 recordings, we believe that Ch1 signals leaked to Ch2 since neurons at same location have similar shape of signals but different intensities (neuron in Ch2 should have a static signal).

I noticed that
- case1: baseline level sof some Ch2 neurons are higher than same neurons in Ch1. E.g., neuron 87 in Ch1 VS neuron 52 in Ch2 as shown in the attached pic.
- case 2: baseline levels of same neurons in Ch1/Ch2 are about the same. E.g., neuron 246 in Ch1 VS neuron 64 in Ch2.

I wonder if these two signals are comparable? Can I come up with a conclusion: signals of Ch2 got pushed up because of the original signals of neuron in Ch2? Thank you for you help!

case 1:

case2:

[kushalkolar]

Are you describing fluorescence bleed-through? If so that's due to your optical setup and/or the indicators you're using. Depending on whether you're using filters, a prism, etc., to collect your light you want to have it set up so you don't get bleed-through.

[XiaoqianSun0104]

Thanks for the reply.
I'm not asking how to deal with or prevent the bleed-through. The contamination is already there but I'm still asked to analyze the data. Let me reframe my question:
- assume Ch1 has 100 neurons (2 groups, group A-50 neurons, group B-50 neuron), GCaMP6m used
- td-Tomato was used to label the nucleus in Ch2 only (Ch2 should have constant fluorescence)
- Ch2 marked group A
- leak from Ch1 contaminated Ch2
- aim: separate group A and group B in Ch1 based on whatever info extracted in Ch2

I plotted estimated.C of 2 pairs of neurons (attached), and can I conclude these two neurons belong to different groups based on differences of the baseline levels between Ch1 and Ch2? Thank you!