Scanning electroporation of selected areas of adherent cell cultures

We present a computer-controlled scanning electroporation method. Adherent cells are electroporated using an electrolyte-filled capillary in contact with an electrode. The capillary can be scanned over a cell culture and locally deliver both an electric field and an electroporation agent to the target area without affecting surrounding cells. The instantaneous size of the targeted area is determined by the dimensions of the capillary. The size and shape of the total electroporated area are defined by these dimensions in combination with the scanning pattern. For example, striped and serpentine patterns of electroporated cells in confluent cultures can be formed. As it is easy to switch between different electroporation agents, the method is suitable for design of cell cultures with complex composition. Finite element method simulations were used to study the spatial distributions of the electric field and the concentration of an electroporation agent, as well as the fluid dynamics related to scanning and flow of electroporation agent from the capillary. The method was validated for transfection by introduction of a 9-base-pair-long randomized oligonucleotide into PC12 cells and a pmaxGFP plasmid coding for green fluorescent protein into CHO and WSS cells

Numerical Calculations of Single-Cell Electroporation with an Electrolyte-Filled Capillary

An electric field is focused on one cell in single-cell electroporation. This enables selective electroporation treatment of the targeted cell without affecting its neighbors. While factors that lead to membrane permeation are the same as in bulk electroporation, quantitative description of the single-cell experiments is more complicated. This is due to the fact that the potential distribution cannot be solved analytically. We present single-cell electroporation with an electrolyte-filled capillary modeled with a finite element method. Potential is calculated in the capillary, the solution surrounding the cell, and the cell. The model enables calculation of the transmembrane potential and the fraction of the cell membrane that is above the critical electroporation potential. Electroporation at several cell-to-tip distances of human lung carcinoma cells (A549) stained with ThioGlo-1 demonstrated membrane permeation at distances shorter than ∼7.0 μm. This agrees well with the model's prediction that a critical transmembrane potential of 250 mV is achieved when the capillary is ∼6.5 μm or closer to the cell. Simulations predict that at short cell-to-tip distances, the transmembrane potential increases significantly while the total area of the cell above the critical potential increases only moderately

Nanopipette Delivery of Individual Molecules to Cellular Compartments for Single-Molecule Fluorescence Tracking

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane