Single yeast cell vacuolar milieu viscosity assessment by fluorescence polarization microscopy with computer image analysis

This study was undertaken to evaluate the apparent viscosity within the vacuoles of single Saccharomyces cerevisiae cells by steady‐state fluorescence anisotropy measurements of quinacrine, using wide‐field fluorescence polarization microscopy combined with computer image analysis. Quinacrine was shown to be rather specifically accumulated within the vacuoles of the cells. This accumulation was effectively reversed by ATP depletion of the cells, with no detectable binding of the dye within the vacuoles. Quinacrine fluorescence anisotropy in the sucrose solutions of various viscosities obeyed the Perrin equation. The fluorescence anisotropy of quinacrine was measured in the vacuoles of 39 cells. From cell to cell, this parameter changed in the range 0.032–0.086. Using the Perrin plot as a calibration curve, apparent viscosity values of the vacuolar milieu were calculated for each cell. The population of the cells studied was heterogeneous with regard to vacuolar viscosity, which was in the range 3.5 ± 0.4–14.06 ± 0.64 cP. There was a characteristic distribution of the frequencies of cells with apparent viscosities within certain limits, and cells with viscosity values in the range 5–6 cP were the most frequent. No relationship was found between the sizes of the vacuoles and their apparent viscosities. Copyright © 2012 John Wiley & Sons, Ltd.