We have developed two tools that can be used to quantify the intensity of one fluorescent marker that overlays another fluorescent marker. We have used these to quantify the buildup of actin filaments on mitochondria, which we refer to as ADA (acute damage-induced actin, described in PMID 35290799). However, these methods can be used to quantify any change in fluorescence in specific areas of the cell (as long as that area is fluorescently labeled itself).
Our programs
There are two different tools, both using plugins we wrote for ImageJ/Fiji. In the descriptions below, we will use the ADA example to describe the intensity of actin on mitochondria, but one can substitute one’s own terms instead.
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1) Live-cell analysis. This set of tools measures the change in actin intensity on mitochondria over time. In our particular example, we collect movies over 10 min, imaging every 15 sec. We stimulate cells at 1 min after the start of image acquisition. The analysis tool output is the change in mitochondrial actin intensity, in which the initial actin intensity (average of the first 4 frames, before stimulation) is normalized to 1.
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2) Fixed-cell analysis. This set of tools measures the difference in actin intensity between mitochondria and non-mitochondria regions. We trace cells (being careful to avoid the peripheral regions which are stress fiber-abundant) and then the tool segments the mitochondria and measures the actin intensity of both the mitochondrial and non-mitochondrial regions. The output is a ratio of mitochondrial:non-mitochondrial actin intensity. In our experience with U2OS and HeLa cells, this ratio is generally 1.1-1.2 for unstimulated cells.
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