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-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.     

Tetragonal distortion field of hydrogen atoms in iron

The difference between the chemical potentialμ _σ of hydrogen atoms producing a nonspherical symmetry strain in a solid sample under stress and that μ₀ corresponding to the state without stress has been calculated. It is shown that μ_σ - μ₀ = -VΣiσ_iε _ii ^′ =U whereσ _i stands for principal stress,ε _ii ^′ for the strain component along the direction of the principal stress,V for volume, andU is the interaction energy between the strain field and external stress field. The hydrogen atoms producing the tetragonally symmetric strain are preferentially ordered in samples under stress. As a result, the variation of hydrogen concentration with tensile stress σ will be different from that with compressive stressσ*. For a general polycrystal the formulas are, respectively,C _ten =C ₀ exp[(0.70089ε₁₁ + 0.2991lε₂₂)Vσ/RT]andC _com =C ₀ exp[(0.14956ε₁₁ + 0.85044ε₂₂)Vσ*/RT], whereC _ten.,C _com., andC ₀ are, respectively, the hydrogen concentrations under tensile stress, compressive stress, and without stress; R stands for the gas constant andT for absolute temperature. Hence, ε₁₁/ε₂₂ may be determined in terms ofC _ten/C _com which can be obtained by hydrogen permeation measurement. For example, according to Bockris' data ε₁₁/ε₂₂ = 1.27 at temperature of 27 °C which implies that the strain field of hydrogen atoms inα-Fe is nonspherical symmetry. For a torsional stressτ,U _tor = -0.55133V _τ(ε₁₁ ε₂₂. The interaction can result in the enrichment of hydrogen atoms on and hydrogen induced delayed cracking along the planes inclined at an angle ofα = 45 deg to torsional axis, which was observed in precharged smooth torsional or type III cracked specimens made of ultra-high strength steel.