Measuring Fluorescent Intensity and the background

Hello everyone,

I’m new to ImageJ and I have no idea what I’m doing!

So far I’ve been trying to work out the fluorescent intensity of Fura-2 at different time points on Image J.

What I’ve been doing: For each Image I’ve been measuring the ROI and averaging the values for each image and then I’ve been subtracting the background, which has been different for each image.

Is it normal to have different background for each image? Am I doing the right thing?
28-08-2019_1_Fl before inc.tif (284.0 KB)
Kind regards,
J

If it is constant decay due to bleaching you might be able to try something like this:

If it is flickering, it could be the focus changing, in which case I’m not as sure what a good option would be.

yeah, if there’s some bleaching of unassociated dye in the area around the cells, it’s normal to see some decay due to bleaching as @Research_Associate pointed out. I don’t think you need to do anything all that fancy to correct it though, just a per-timepoint background subtraction, as it sounds like you’re doing. hard to say for sure from the single timepoint image you uploaded though.
Regarding focal drift, I wouldn’t expect that to have a huge impact on the background intensity unless it’s very dramatic drift (is this widefield?). Also, the ratiometric nature of Fura-2 should help minimize the impact of focal drift. Are you collecting ratiometric images? or just a single excitation wavelength? (the image you uploaded seemed like a single channel).