This is a simple common method for ratio FRET with CFP-YFP biosensor imaged with a 442nm laser using a Zeiss 880 confocal with a 60X N.A. 1.4 lens.

simple ratio macro

Here is another example with a graph, however, the increased ratio is due to bleaching of one fluorescent protein at a different rate than the other. This was an early try and settings had to be changed to avoid the bleaching issue. Also, bleaching curves may be characterized and included in the formula for calculating the ratio, but this requires standard settings and cells with similar expression levels of FPs.


The example below is more similar to how this might be done in bulk with a spectrophotometer.