Visualization of membrane loss during the shrinkage of giant vesicles under electropulsation

We study the effect of permeabilizing electric fields applied to two different types of giant unilamellar vesicles, the first formed from EggPC lipids and the second formed from DOPC lipids. Experiments on vesicles of both lipid types show a decrease in vesicle radius which is interpreted as being due to lipid loss during the permeabilization process. We show that the decrease in size can be qualitatively explained as a loss of lipid area which is proportional to the area of the vesicle which is permeabilized. Three possible mechanisms responsible for lipid loss were directly observed: pore formation, vesicle formation and tubule formation.Comment: Final published versio

Apoptosis- and necrosis-induced changes in light attenuation measured by optical coherence tomography

Optical coherence tomography (OCT) was used to determine optical properties of pelleted human fibroblasts in which necrosis or apoptosis had been induced. We analysed the OCT data, including both the scattering properties of the medium and the axial point spread function of the OCT system. The optical attenuation coefficient in necrotic cells decreased from 2.2 ± 0.3 mm−1 to 1.3 ± 0.6 mm−1, whereas, in the apoptotic cells, an increase to 6.4 ± 1.7 mm−1 was observed. The results from cultured cells, as presented in this study, indicate the ability of OCT to detect and differentiate between viable, apoptotic, and necrotic cells, based on their attenuation coefficient. This functional supplement to high-resolution OCT imaging can be of great clinical benefit, enabling on-line monitoring of tissues, e.g. for feedback in cancer treatment

Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts

Studies of cell pellets: I. Electrical properties and porosity.

Cell pellets formed by centrifugation provided a good system to study the osmotic behavior, electroporation, and interaction between cells. Rabbit erythrocyte pellets were used in this study because they were simpler than nucleated cells to model analytically. Structurally, cell pellets possessed properties of porous solid bodies and gels. Electrically, cell pellets were shown to behave as a parallel set of resistance, Rp, and capacitance, Cp. Information on pellet structures was obtained from electric measurements. The pellet resistance reflected the intercellular conductivity (porosity and gap conductivity), whereas the pellet capacitance depended mostly on membrane capacitance. The pellet resistance was more sensitive to experimental conditions. The intercellular gap distance can be derived from pellet porosity measurements, providing the cell volume and surface area were known. Rp increased and relaxed exponentially with time when centrifugation started and stopped; the cycles were reversible. When supernatants were exchanged with solutions containing hypotonic electrolytes or macromolecules (such as PEG) after the pellets were formed, complicated responses to different colloidal osmotic effects were observed. A transient decrease followed by a large increase of Rp was observed after the application of a porating electric pulse, as expected from a momentary membrane breakdown, followed by a limited colloidal-osmotic swelling of pelleted cells. The equilibrium values of Rp, Cp, pellet porosity, and intercellular distances were measured and calculated as functions of cell number, centrifugation force, and ionic strength of the exchanged supernatant. Thus, the structure and properties of cell pellets can be completely characterized by electrical measurements

Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions.

Using the relations between pellet structure and electric properties derived from the preceding paper, the responses of rabbit erythrocyte pellets to osmotic or colloidal-osmotic effects from exchanged supernatants and from electroporation were investigated. Changing the ionic strength of the supernatant, or replacing it with dextran or poly(ethylene glycol) solutions, caused changes of Rp according to the osmotic behavior of the pellet. Rp was high and ohmic before electroporation, but dropped abruptly in the first few microseconds once the transmembrane voltage exceeded the membrane breakdown potential. After the initial drop, Rp increased as a result of the reduction of intercellular space. Rp increased regardless of whether the pellets were formed before or immediately after the pulse, indicating that porated cells experienced a slow colloidal-osmotic swelling. The intercellular or intermembrane distances between cells in a pellet, as a function of osmotic, colloidal-osmotic, and centrifugal pressures used to compress rabbit erythrocyte pellets, were deduced from the Rp measurement. This offered a unique opportunity to measure the intermembrane repulsive force in a disordered system including living cells. Electrohemolysis of pelleted cells was reduced because of limited swelling by the compactness of the pellet. Electrofusion was observed when the applied voltage per pellet membrane exceeded the breakdown voltage. The fusion yield was independent of pulse length greater than 10 microseconds, because after the breakdown of membrane resistance, voltage drop across the pellet became insignificant. Replacing the supernatant with poly(ethylene glycol) or dextran solutions, or coating pellets with unporated cell layers reduced the colloidal-osmotic swelling and hemolysis, but also reduced the electrofusion yield. These manipulations can be explored to increase electroloading and electrofusion efficiencies