Determination of rotational diffusion coefficients

When the excitation light is polarized and/or if the emitted fluorescence is detected through a polarizer, rotational motion of a fluorophore causes fluctuations in fluorescence intensity. We will consider only the case where the fluorescence decay, the rotational motion and the translational diffusion are well separated in time. In other words, the relevant parameters are such that tc rp, where is the lifetime of the singlet excited state, zc is the rotational correlation time (defined as l/6Dr where Dr is the rotational diffusion coefficient see Chapter 5, Section 5.6.1), and td is the diffusion time defined above. Then, the normalized autocorrelation function can be written as (Rigler et al., 1993) 

This relation shows that the rotational correlation time is uncoupled from the excited-state lifetime, in contrast to classical steady-state or time-resolved fluorescence polarization measurements (see Chapter 5). The important consequence is the possibility of observing slow rotations with fluorophores of short lifetime. This is the case for biological macromolecules labeled with fluorophores (e.g. rhodamine) whose lifetime is of a few nanoseconds. 

Note that the translational diffusion time decreases when the beam radius is decreased but not the rotational correlation time. 

---