Cytoplasmic viscosity near the cell plasma membrane: measurement by evanescent field frequency-domain microfluorimetry

Abstract

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-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser 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 monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.

Intracellular Trafficking of Plasmids for Gene Therapy: Mechanisms of Cytoplasmic Movement and Nuclear Import

“…It had previously been shown by spotphotobleaching that small solutes could diffuse freely and rapidly in the cytoplasm and the nucleus [Bicknese et al 1993;Kao et al 1993]. This suggested that diffusion of large macromolecules like DNA may be possible in the cytoplasm.…”

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis

“…Most methods for intracellular viscosity measurements are based on the use of fluorescent (Luby-Phelps et al, 1993;Bicknese et al, 1993) or spin probes (Morse, 1986). Loading of live (unfixed) yeast cells, and specifically their vacuoles, with the probes remains a problem as yet.…”

Influence of semiflexible structural features of actin cytoskeleton on cell stiffness based on actin microstructural modeling

“…The stiffness k c of the ABPs is set at 100 pN/mm, consistent with the parameters (100-10,000 pN/mm) reported in literature (Lang et al, 1986;Molloy et al, 1995;Minoura et al, 1999;Bicknese et al, 1993;Shafrir and Forgacs,(2002). The actin filaments and actin-binding proteins are immersed in the cytosol.…”