I’ve imaged the cells as z stack for two channels (Green and Red) with different settings because the brightness of green was very low. And I want to do ratiometric measurements, (green intensities/Red intensities). What is the best method to calculate overcome the difference in the illumination settings?
And what is the best method to do z stack intensity measurement?
Since you are looking at the ratio image, the FRET experiment may be helpful. (IMD, etc.).
Also, there have been several threads on FRET in the past, so those may be helpful as well.
Even if the brightness settings are different, there is no problem in comparing within the same image using the ratio.
However, it is necessary to pay attention to the Z projected image of the ratio image.
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Just to clarify, since we get people from all sorts of backgrounds here, if you are exciting with two different light sources/intensities, there is no real meaning to the ratio you are calculating other than in references to other ratios calculated with the exact same settings.
IE, without some sort of known concentration control, you will never know the actual relative abundance of your two channels. Just the relative relative abundance, so you can tell if the ratio is shifting.
That said, background subtraction is probably going to be very important, and if you have a Z stack, you may want to look into volume based sums. However, it is important how you use the background subtraction. For example, background from the media would not show up inside the cell, so subtracting it from that volume would be incorrect. On the otherhand, background from the camera would likely show up everywhere.
Even then, it is usually best to have some sort of third channel, whether it be brightfield or blue or far red to determine the borders of the regions you want to measure. Otherwise, a cell that is “dim” for both of your markers may appear smaller, or not show up at all after thresholding. Whenever possible measurements should be made inside of a volume determined by something other than the measurement channel itself.
There are quite a few posts on general Z-stack analysis, here is one.
Thank you very much for your detailesd informations.
I am doing the expermints using pH nanosensors, I’ve prepared control spheroid without any condition to know the real intensity. And I’ve added different drug concentrations to show change in pH. Did you mean that by control?
Hard to say without knowing more about your experiment. Just that generally, intensity based measurements are relative only, unless you can take them on a sample with a known concentration/whatever.
Not sure how your nanosensors work as it could be FLIM, FRET, or something else. If it is pure intensity ratios, that seems risky, but I am not an expert in pH measurements and don’t have the details of your setup. Just be careful