Good thing takes time

Polarized light can excite the fluorophore with the same orientation, aligned.

For measurement:
P=(Iv-Ih)\(Iv+Ih)
r=(Iv-Ih)\(Iv+2Ih)
P=3r\(2+r)

For physical meaning:
(1\P-1\3)=(1\P0-1\3)(1+Life time of fluorophore\rotation correlation time)

P is positively correlated with rotation correlation time
rotation correlation time=viscoicty*Mw*(specific volume+hydration)\RT

hydration=0.2 g water per gram of protein

For application:

fluorescein: life time is 4 ns, used for Mw less than few kilodalton (kDa).
Metal-ligand complex (Ru, Os, Re): life time is few hundred ns to millisecond, for Mw higher than 10 kilodalton