Phasor approach for fluorescence anisotropy

Fluorescence anisotropy is a powerful tool for observing molecular rotational speed, which is widely applied for molecular conjugation measurements (quantitative assay, membrane tagging, and protein-protein interaction). Fluorescence anisotropy (r) can be obtained by subtracting horizontal polarized fluorescence (H) from vertical polarized fluorescence (V), and dividing by non-polarized fluorescence (F). Since F equals to V+2H, V and H can be thought of as weighted sum and subtraction of F and r·F, respectively. Using phasor approach, which is graphical plotting technique based on fluorescence lifetime imaging microscopy, V and H are located on internal and external dividing points of F and r·F. Considering those four phasor points (V, H, F, and r·F) lie on a straight line, one can easily guess the value of the r. We first introduced phasor plot to fluorescence anisotropy, and confirmed that this method dramatically simplifies fluorescence anisotropy analysis with graphical intuition.