R' method for determination of parameter alpha for FRET

Parameter alpha is required for the calculation of the energy transfer efficiency in intensity-based FRET experiments. We developed a method for determining alpha in microscopic implementations of intensity-based FRET measurements using a single sample. This approach requires a parameter, R', that describes the fraction of binding sites occupied by the donor antibodies in cells or beads labeled with a mixture of donor-tagged and acceptor-tagged antibodies, and it is also influenced by the degrees of labeling of the two antibodies.