title: 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.
authors.affiliation: Department of Medicine, University of California, San Francisco 94143-0532.
authors.forename: S
authors.initials: S
authors.lastname: Bicknese
authors.forename: N
authors.initials: N
authors.lastname: Periasamy
authors.forename: S B
authors.initials: SB
authors.lastname: Shohet
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003592:Cytophotometry / Q000295:instrumentation; D003593:Cytoplasm / Q000378:metabolism*; D004285:Dogs; D005452:Fluoresceins; D005454:Fluorescence Polarization; D051379:Mice; D008962:Models, Theoretical; D012996:Solutions; D014783:Viscosity; D014867:Water
chemical_list: D005452:Fluoresceins; D012996:Solutions; D014867:Water; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein