CYTOPLASMIC VISCOSITY NEAR THE CELL PLASMA-MEMBRANE - MEASUREMENT BY EVANESCENT FIELD FREQUENCY-DOMAIN MICROFLUORIMETRY

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethy l)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transf orm microfluorimetry (Fushimi and Verkman, 1991). Information about vi scosity within <200 nm of cell plasma membranes was obtained by select ive excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized lase r illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and m onolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canin e kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theor etically and/or experimentally, including variations in lifetime due t o fluorophore proximity to the interface, and the use of s and p polar ized excitation. In fluorescein solutions excited with s-plane polariz ed light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotatio nal correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Ap parent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly diff erent from the viscosity measured in bulk cytoplasm far from the plasm a membrane. The results establish the methodology for time-resolved mi crofluorimetric measurement of polarization in the evanescent field an d demonstrate that the cell plasma membrane has little effect on the f luid-phase viscosity of adjacent cytoplasm.