Fluorescence polarization spectroscopy at combined high-aperture excitation and detection: application to one-photon-excitation fluorescence microscopy.

A problem of the one-photon-excitation fluorescence polarization spectroscopy of macroscopically isotropic media, in the case of combined high-aperture excitation and detection, is considered and described in a spherical representation. The case of inhomogeneous intensity distribution in the cross-section of the parallel beam of exciting light, which is converted by an objective lens into inhomogeneous radial distribution of the intensity of the focused exciting light, is also taken into account. The obtained formalism is adapted to the description of confocal fluorescence polarization microscopy. It is shown that the total and magic-angle-detected fluorescence decays do not solely represent the kinetic evolution of the excited-state because of the contribution of the dynamic evolution of photoselected fluorophores. The time-evolution of emission anisotropy is nonexponential. The outlined theory predicts that the total and magic-angle-detected fluorescence decays solely represent the kinetic fluorescence decay, and thereby, the emission anisotropy becomes an (multi)exponential function of time for the excitation-detection cone half-angles not higher than about 15-20 degrees. The initial values of the emission anisotropy are not modified by the application of the excitation-detection apertures if the cone half-angles do not exceed 10-15 degrees. The histograms of unpolarized fluorescence, calculated from the parallel and the perpendicular components of polarized fluorescence, detected at the excitation-detection cones wider than about 65 degrees solely represent the kinetic fluorescence decay. At such conditions, the microscope objective operates like an "integrating sphere". The calibration method, which is based on a general (symmetry adapted) formula describing fluorescence polarization experiments on macroscopically isotropic samples, is discussed. This method enables the analysis of all fluorescence polarization experiments without the necessity of considering the expressions for polarized fluorescence decays relating to a particular experimental case of interest. With this method, any commercially available microscope objective can be calibrated, and its optical properties can be precisely verified. The application of the outlined theory to different fluorescence spectroscopy techniques is indicated. The expressions derived for confocal fluorescence polarization microscopy can be employed in the numerical analysis of the data recovered from the photochemical bioimaging.