Flow cytometric measurement of the polarization of fluorescence from intracellular fluorescein in mammalian cells

Abstract

Based on the description of a laboratory-built flow cytometer, the necessary modifications of this instrument for the measurement of fluorescence polarization are described. At a maximum rate exceeding 1,000 cells/s, the instrument is capable of measuring simultaneously the horizontally and vertically polarized component of the fluorescence emitted from stained cells excited with vertically polarized light. By mathematical analysis of the accumulated data, the distribution of polarization values in the population is obtained. Various sources of instrumental error have been investigated. The large aperture of the detector optics leads to systematic underestimation of the polarization values. Other errors are negligible, and the instrument is shown to give results consistent with the theory of fluorescence polarization. Application of the instrument is illustrated by experiments with mammalian cells exposed to the fluorogenic substrate fluorescein diacetate (FDA). The polarization of the fluorescence from intracellular fluorescein produced by hydrolysis of FDA is measured, giving information on the cytoplasmic microviscosity. It appears that this microviscosity is constant over the cell cycle. On the other hand, it is significantly affected by the osmolarity of the medium.