Spectral imaging of time-resolved anisotropy: theory and experiment

Time-resolved fluorescence anisotropy on the nanosecond time scale is useful for the study of the rapid rotation of macromolecules. A system combining the capabilities of fluorescence spectral imaging with time-resolved fluorescence anisotropy and enabling the wide-field measurement of the spectroscopic parameters of fluorophores is discussed. The phasor approach is used to quantitatively analyze the time-resolved fluorescence anisotropy by transforming the polarized parallel and perpendicular components to the phasor space in the frequency domain, respectively, and a unique way to calculate the fluorescence rotational correlation time is put forward. Experimental results prove that the phasor approach is a proper model for the time-resolved fluorescence anisotropy.