Trapping, deformation, and rotation of giant unilamellar vesicles in octode dielectrophoretic field cages

Abstract

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy.

FIGURE 1

Dielectrophoretic responses and electrode phase assignments in microfield cages. (A) Real (d) and imaginary (r) part of the normalized complex polarizability (Clausius-Mossotti-factor) for GUVs. Since GUVs are not small compared to cage dimensions, quadrupole contributions are also shown (index q). GUVs were modeled as shelled spheres with the following parameters: diameter, 20 μm; shell thickness, 6 nm; shell permittivity, 8; shell conductivity, 1 μS/m; medium (internal) conductivity, 0.69 S/m (50 mM KCl); and medium permittivity, 78. A typical surface of constant mean-square electric field is shown for (B) ac and (C) rot electrode assignment modes.

FIGURE 2

Trapping and electrorotation of GUVs and particles in the dielectric microfield cage. (A) Confocal image of the equatorial plane of a single trapped GUV, labeled with diI-C_20:0. (B) Corresponding electrode outline and dielectric field potential in the central horizontal plane in units of the applied mean-square electric field. The difference between two contour lines corresponds to 2%. The dielectrophoretic response of latex particles is shown in C. The second column (D–F) shows images for rotating mode. The electrode voltage and frequency assignments were 4.4 V_rms and 0.75 MHz, respectively. The distance between opposing electrodes is 40 μm.

FIGURE 3

Three-dimensional image reconstructions of GUVs trapped and deformed in octode field cages. In each panel, the left image shows a confocal micrograph of the equatorial region, the central image shows the top-view projection of the upper half of the GUV, and the right image a side-view projection. GUVs shown in E and F were made from DPPC only. Field conditions were (A, B, E, and G) 1.8 V_rms, 0.187 MHz; (C) 2.5 V_rms, 0.75 MHz; (F) 4.4 V_rms, 0.187 MHz, all ac-mode; and (D) 2.5 V_rms, 0.187 MHz, rot-mode.

FIGURE 4

Voltage- and frequency-dependence on vesicle shape and brightness of diI-C_20:0 in octopole field-cage trapped GUVs (ac-mode). (A) Voltage series at constant frequency of 0.75 MHz. The value of 1.8 V_rms is shown twice to demonstrate the reversibility of the fluorescence brightness change. (B) Frequency dependence at a constant voltage of 4.4 V_rms. The average membrane fluorescence intensity per pixel, measured across the entire membrane circumference, is given on the top right of each image as an 8-bit gray value.

FIGURE 5

FCS on GUVs trapped in octode field cages. (A) FCS curves of diI-C_20:0 in the absence (solid line) and presence (dashed line) of an electric field in the cage (3.5 V_rms, 2.9 MHz, ac-mode). (B) Diffusion coefficients as a function of electric field, determined from fits to FCS curves as shown in A to a model of two-dimensional translational diffusion. The 0 V data point was measured on the GUV after it had settled to the bottom wall of the chamber. The error on the determined diffusion coefficients was ∼20%.

FIGURE 6

Hydrodynamic streaming as a function of electric field inside the octode cage, measured by FCS of 100-nm latex beads. The relative position of the focal volume with respect to the electrodes is indicated schematically: between the upper electrodes for A and B, cage center for C and D. The concentration of KCl was 50 mM (0.65 S/m) in A and C, and 12.5 mM (0.16 S/m) in B and D. In all cases, the frequency was 0.75 MHz, ac-mode. E shows fluid velocities determined from fitting the FCS curves for the four conditions.