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Polarization-resolved fluorescence lifetime imaging (FLIM) can provide information about the rotational diffusion of a fluorophore. We have developed a wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM) system to quantitatively image both the fluorescence lifetime and the rotational correlation time of a fluorophore. Upon excitation with linearly polarized light, we simultaneously image the parallel and perpendicular polarization components of the fluorescence emission employing a polarization-resolved imager in front of a time-gated intensified CCD. We demonstrate this technique by imaging the rotational correlation time of fluorescein in solutions of varying viscosity in a multiwell plate and in live cells. Introduction

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore