Supplementary Materials and Methods: PEF Dosimetry for ITO coverslips

1. Simulation of the electric field distribution

The simulation model of the electroporation cuvette with the integrated glass/ITO structure has been implemented in COMSOL Multiphysics Software version 4.4 (Burlington, USA) environment.

Because the glass/ITO structure features a cylindrical shape, the model has been approximated as a three-layer axisymmetric 3D geometry, forming a cluster structure, which consists of a buffer solution between the cuvette electrodes, the glass substrate with the thickness of 150 μm and the radius of 4 mm, covered with a 120-nm thick ITO layer (Fig. S1).

To simulate the distribution of the strength of the electric field created within the cuvette by a high voltage electric pulse applied to the electrodes of a cuvette, the glass/ITO layer was positioned on the high-potential (+100 V) positive electrode (anode), while the ground terminal was connected to the opposite metallic surface (cathode). These electrodes acted as voltage sources and appropriate boundary conditions were established for their surfaces. Subsequently, the system of an axisymmetric geometry was divided into finite elements. The maximum element size was 0.15 mm and the minimum was 0.1 μm, respectively. A maximum element growth rate of 2 with the curvature factor of 0.2 was selected, resulting in a finished mesh structure, which featured 158,627 (51539 domain and 5964 boundary) elements. The complete list of the parameters used for the simulation of the applied model is presented in Table S1.

The layer of adherent cells on the ITO surface was not included into the system model. Thus, the influence of cells on the distribution of the electric field was not evaluated.

The results of the simulations, for a 1-mm gap cuvette, are presented in Fig. S2. Panel (a) shows a top view (x-y cross-section) of the distribution of the electric field strength at the surface of the ITO layer (at the height of 150.2 m from the surface of the underlying electrode of the electroporation cuvette). Panel (b) shows the electric field in the x-z cross-section through the center of the glass coverslip, and panel (c) is a graph of the electric field strength in the same x-z cross-section, at 150.2 m m above the underlying electrode.

This figure demonstrates that the electric field is lower above the central part of the ITO surface compared to the regions “outside” the coverslip. The electric field strength above the central part of the ITO surface equals 0.28 kV/cm, or 28% of 1.0 kV/cm electric field produced by applying 100 V over a 1-mm gap in the absence of the coverslip. This proportionality coefficient does not depend on the pulse duration or the voltage applied to the cuvette. Similarly calculated electric field attenuation coefficients for 2- and 4-mm gap cuvettes were 50% and

Fig. S1. A mesh of finite elements for the model of a glass coverslip covered with ITO layer within the electroporation cuvette. The glass coverslip with the radius of 8 mm is positioned within the electroporation cuvette filled with the electroporation medium on the high-potential (+100 V) positive electrode (anode). The distance between the electrodes is 1 mm.

65%, respectively. Although 2- and 4-mm gap cuvettes were not used for biological experiments, these simulation results were essential for verification of the accuracy of the simulations by experimental measurements (see below).

Table S1. Parameters for simulation of the electric field distribution

Parameter Value Denotation

Voltage applied between the electrodes, V 100 UAP

Distance between the electrodes, mm 1.0 g

Thickness of the ITO layer, m 0.12 w1

Thickness of the glass coverslip, mm 0.15 w2

Radius of a glass coverslip, mm 4.0 rITO

Relative permittivity of glass 2.3–4.6 εG

Relative permittivity of ITO 9.3 εITO

Relative permittivity of the cell culture medium (water) 77–80 εM

Specific conductivity of glass, S/m 10-14 σG

Specific conductivity of ITO, S/m 4.6×104–2.2×105 σITO

Specific conductivity of the exposure medium, S/m 1.2–1.8 σM

The electric field is homogeneous (>90%) within the circle with approximate radius of 3.3 mm. There is a gradual reduction further outward this region with an increase of the field

Fig. S2. Electric field at the surface of an 8-mm ITO coverslip placed into a 1-mm gap electroporation cuvette. The cuvette is filled with the exposure medium; the coverslip is placed flat on the positive electrode (anode) with ITO surface facing up (into the medium); 100 V is applied between the walls of the cuvette. (a) A top view (x-y cross-section) of the distribution of the electric field at the surface of the ITO layer (1 m above it). (b) A cross-sectional view (x-z cross-section) of the distribution of the electric field within the cuvette, with the section taken through the center of the glass coverslip. (c) The electric field at the height of 150.2 m above the anode (i.e., at the surface of the ITO coverslip), in the x-z plane going through the center of the coverslip.

strength within a narrow (0.1-0.2 mm) outer rim and right outside the coverslip. This is because regions of the local strong electric field are created at the sharp edges of a thin conductive ITO layer.

The decrease and the increase of the electric field at the very periphery of the ITO layer had little or no impact on the measured cell survival. First, these areas were small compared to the central region with a uniform electric field. Second, the way cells were seeded on the coverslips (by placing a large drop of cell suspension atop the ITO and leaving it undisturbed until cells precipitate and attach) resulted in a higher cell density in the central areas whereas relatively few cells attached at the peripheral rim. Nonetheless, the potential non-uniformity of the electric field was taken into account when analyzing biological data, especially when isolated surviving cells were found at high exposure doses.

2. Experimental measurements of the electric field on ITO surface

These measurements were undertaken to verify the simulation predictions. A tungsten microelectrode (World Precision Instruments, Sarasota, FL) was glued with a drop of electroconductive silver glue to the center of the ITO layer. The microelectrode shaft was 0.1 mm in diameter, and it was electrically insulated except for the glued tip. The electrode with the glued coverslip was fixed in a micromanipulator to position the coverslip within an electroporation cuvette, with its non-ITO surface touching flat against the anode electrode (Fig S3).

Pulses of 300- or 600-ns duration, at various amplitudes from 300 to 900 V, were delivered from Avtech AVOZ-D2-B-ODA generator. The resulting electric potential at the center of the ITO surface in respect to the ground electrode (cathode) was measured. The measurement results matched well with those predicted by simulations (Table S2). Small differences of the measured and simulated values can be explained by the presence of the measurement microelectrode (which affected the current density and the uniformity of the electric field), as well as by deviations of the actual cuvette gap from the nominal value (up to 20% for 1 mm cuvettes, data not shown). The impact of both confounding factors expectedly decreased for larger gap cuvettes.

Table S2. Comparison of measured and simulated electric potential values at the surface of ITO coverslip inserted in an electroporation cuvette. The coverslip was placed on the positive electrode, and the potential was measured between the ITO center and the opposite wall of the cuvette (negative electrode).

Cuvette gap (mm)

Applied voltage (V)

Measured ITO potential (V)

Simulated ITO potential*, V

Difference between the measured and simulated values, % of the applied voltage

1 410 132 98 +8.31 923 264 220 +4.82 400 162 185 -5.8

Fig. S3. An ITO coverslip placed into a 1-mm gap electroporation cuvette for measurements of the electric potential at the ITO surface (not to scale). A metal microelectrode is glued to the ITO surface. See text for more details.

2 500 240 231 +1.82 900 368 416 -5.34 516 332 323 +1.84 412 262 258 +1.0

* The simulated ITO potential (Vsim) was calculated as: Vsim = (k x Va) x (d/g), where k is a proportionality coefficient from electric field simulations (0.28, 0.5, or 0.65 for 1-, 2- and 4-mm gap cuvettes, respectively); Va is the applied voltage, (V); d is the distance from the ITO surface to the opposite wall of the cuvette; and g is the nominal gap of the electroporation cuvette. In all cases, the difference between d and g was 0.15 mm (the thickness of the coverslip).