Cell type and spatial location dependence of cytoplasmic viscosity measured by time-resolved fluorescence microscopy

Abstract

Information on the cell type and spatial location dependence of cytoplasmic viscosity would be very useful in understanding some of the processes occuring in the cell. For this purpose, fluorescent dye kiton red (sulforhodamine B) was loaded into a variety of cells such as Swiss 3T3 fibroblasts, human mononuclear cells, Sarcoma-180 tumor cells, Chinese hamster ovary cells, plant cells fromDigitalis lanata,stamen hair cells ofTradescantia,and guard mother cells ofAllium cepa.Space-resolved measurements of cytoplasmic viscosity were carried out by using an experimental set-up wherein a picosecond laser system was coupled with an epifluorescence microscope. The spatial resolution of this set-up was ~1.0 μm, and reliable dynamic fluorescence measurements could be obtained from 10² to 10³ fluorescent molecules. Fluorescence lifetime measurements showed that a large fraction (~70%) of kiton red was in the free form. Fluorescence anisotropy decay of kiton red in cells was analyzed by a two population (free and bound) model. The microviscosity of cytoplasm was estimated from the anisotropy decay kinetics of the free probe. It was found that the cytoplasmic viscosity is dependent on both the cell type and spatial location within a cell. Furthermore, both the average value of viscosity and spatial variation within a cell were larger in the plant cells when compared to the animal cells. Model studies in various simpler systems have shown that the higher viscosity observed in some part of the cell could be due to either physical restriction and/or the presence of high concentrations of small solutes and macromolecules.