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Behrend et al.

: Pulse Generators for Pulsed Electric Field Exposure of Biological Cells and Tissues

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Pulse Generators for Pulsed Electric Field Exposure of


Biological Cells and Tissues
Matthew Behrend, Andras Kuthi, Xianyue Gu, P. Thomas Vernier, Laura Marcu,
Cheryl M. Craft and Martin A. Gundersen
Department of Electrical Engineering - Electrophysics
University of Southern California
3737 Watt Way Rm 604
Los Angeles, CA 90089-0271, USA

ABSTRACT
This paper describes three pulse generators: a spark gap switched coaxial cable, a
spark gap switched Blumlein, and a solid state modulator, developed for applying
ultrashort electrical pulses to biological materials in culture. Recent research has
shown that ultrashort pulsed electric fields can induce apoptosis in biological cells,
rm cause
and that pulses as short as 10 ns with field amplitude greater than 1 MVr
membrane phospholipid rearrangement and activation of the effector enzymes of
apoptosis. Pulses of very short duration use only tens of mJ per mL per pulse to
induce apoptosis and other intracellular effects without causing thermal trauma.
The pulse generators discussed here, each of a different topology, deliver ns pulsed
electric fields (nsPEF) to cells in liquid suspension, and can be modified to drive
electrodes for external, surgical, or endoscopic treatment of tissues in situ.
Index Terms Apoptosis, Blumlein, electric field, electroperturbation, fluorescence microscopy, nanosecond, nsPEF, phospholipid inversion, pulsed electric
field, pulse forming network, pulse generator, real-time, ultrashort

INTRODUCTION

ULSE generators for applying nanosecond pulsed


electric fields nsPEF. to biological cells must interface
to interchangeable sterile load chambers and offer variability of pulse parameters as experimental variables in
addition to operating within the correct electrical
impedance matching and pulse shape constraints. Furthermore, pulse generators must be compatible with the
observational techniques employed to assess the cell response to pulsed electric fields. Exposure chambers take
the form of cuvettes and microscope slides. Cuvettes hold
a larger volume to collect response data from millions of
cells. Microscope slides offer real time images of fluorescent tags and morphological changes in observation of a
few to a few hundred cells. In the design of any exposure
chamber, impedance matching and rapid load replacement are the most significant challenges. Three pulse generator designs are compared for their suitability to nsPEF
applications. A single transmission line pulse forming network named the medipulser and the water Blumlein
system named the minipulser have been built to apply
nsPEF to cells in cuvettes. The micropulser is a solid

Manuscript receied on 21 January 2003, in final form 8 May 2003.

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state MOSFET pulse generator for applying nsPEF to cells


on microscope slides.

CELL CONTAINMENT AND


INTERFACE
2.1

CUVETTES

Cells are contained in cuvettes or on a microscope slide


during typical pulse exposures. Cuvettes are convenient
for nsPEF exposure of small volumes of cells less than 1
mL. Cuvettes used by the USC Pulsed Power Group hold
a 75 L volume of cells between opposing flat aluminum
electrodes spaced 1 mm apart. The described cuvette with
cell suspension in RPMI 1640 growth medium presents an
electrical load of 11 in parallel with 55 pF, so pulse
generators for these cuvettes are matched to approximately 10 loads. Standard cuvettes are also commonly
available with 2 mm and 4 mm electrode spacing, having
volumes of up to 0.8 mL.
The cuvette receptacle Figure 1. of the medipulser
pulse generator facilitates quick swapping of cuvettes, important in time-critical biological experiments because
separate cuvettes are used for each sample of cells exposed to nsPEF. The cuvette chamber is a machined

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Figure 1. Cuvette chamber of the medipulser. Spark gap is located


top and center. Pulse input connector is in the upper right.

Teflon housing containing two contact plates that are


pressure mated to the cuvette electrodes. Cuvettes are inserted in the vertical orientation by firmly pressing them
into the side-loading receptacle.
Inductance introduced by the joining of the chamber to
the transmission line is kept low by integrating the spark
gap into the assembly and making the current path area
as tight as possible. The entire cuvette chamber assembly
inserts 22 nH of inductance in series with the cuvette and
the pulse forming transmission line. PSPICE circuit simulation of the medipulser shows a limit on the minimum
pulse length of about 4 ns and a minimum rise time of 2
ns, assuming a transmission line less than 1 ns long. The
minipulser Blumlein pulse generator is less hindered by
series inductance because its dimensions are suited for attachment to cuvettes. However, it also suffers similar lower
limits on pulse dimensions because the cuvette and the
spark gap are both located at the ends of the Blumlein,
external to the dielectric medium. The water Blumlein can
be further improved by placing the spark gap electrodes
inside the dielectric medium, thereby eliminating all inductance associated with the spark gap element. Fast rise
times of 2 ns can be achieved in such a system w3x without
overvoltage operation of the spark gap, but the water spark
gap has a 1 ms recovery time that limits repetition rate.

2.2

MICROSCOPE SLIDES

Cells can be shocked directly on a microscope slide while


being viewed under a fluorescence microscope in quantities of several cells to several hundred cells. The micropulser pulse generator drives an exposure chamber
formed by two adjoining platinum foil strips paraffin
bonded to the surface of a glass slide, having only a narrow gap of 100 m separating the neighboring foil edges.
With a small 100 m chamber, only 400 V is needed to
set up a sufficient nsPEF intensity of 4 MVrm. The relatively low pulse voltage allows high voltage MOSFETs to

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Figure 2. The micropulser mounted to stage insert plate black..


MOSFET gate drive circuit is the daughter board.

serve as the switching element. The nsPEF exposure of


cells on a microscope slide requires the pulse generator to
be integrated with the microscope stage or else to have a
transmission line to carry the pulse to the slide from an
external pulse generator. We chose to build the micropulser onto the stage insert plate for the Zeiss Axiovert 200M fluorescence microscope. The micropulser is
a direct-drive MOSFET pulse generator with the driver
circuit and energy storage capacitor built into an interchangeable stage insert plate Figure 2.. Direct MOSFET
switching eliminates the need for matching a transmission
line to the load and to an external pulse source. The load
only needs to be of higher impedance than the on-state
resistance of the MOSFET. The integrated format was
also chosen to move toward smaller and lower power pulse
generator systems.

PULSE GENERATOR TOPOLOGIES

The choice of topology is directed by the load


impedance, physical form of load e.g. cuvette or slide.,
pulse amplitude, pulse width, rise time, and the desired
variability of pulse parameters, summarized in Table 1.
Transmission line pulse generators are generally not flexible in variability of pulse parameters, but they are suited
for high voltages and fast switching. Two forms of sparkgap-switched transmission lines are shown here to drive
the cuvette loads. Transmission line pulse forming net-

Table 1. Comparison of three pulse generator topologies.


Coaxial Line Water Blumlein Solid State
Max Voltage
Switching Speed
Load Impedance
Charging Source
Pulse Width
Size
Interface to Load

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tens of kV
-1 ns
Zo
2Vload
pulsed
)1 ns
medium
difficult

tens of kV
-1 ns
2Zo
Vload
pulsed
)1 ns
compact
easy

1kV
3ns
4Rds-on
Vload
DC
)12 ns
miniature
easy

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Behrend et al.: Pulse Generators for Pulsed Electric Field Exposure of Biological Cells and Tissues

Figure 3. Single ended transmission line pulse forming network


having characteristic impedance Zo and transmission delay To.

Figure 4. Four RG8X cables bundled to form the medipulser transmission line.

works were selected because of their simplicity and ability


to hold off the kilovolt potentials required for producing
electric fields )1 MVrm in the cuvette. Spark gap switches
are used for their fast ;1 nanosecond switching time.
The pulse-forming network for the medipulser, our single-ended transmission line pulse generator Figure 3., is
a 12 transmission line composed of four 50 coaxial
cables in parallel Figure 4.. The minipulser pulse generator for cuvettes uses a water Blumlein Figure 5. matched
for a 20 load. Transmission line pulse generators require a pulse generator or other high voltage source to
charge them before they can be switched to deliver a pulse
to the load. The charging source may take many forms. A
Blumlein is typically advantageous because the pulse delivered to the load has the same amplitude as the charging
voltage to the line and reduces the design burden for the
charger. However, single ended transmission lines deliver
pulses equal to 1r2 of the charging voltage.
The single ended transmission line topology was selected for the medipulser because the readily available
charging source was not originally intended to drive lower
impedance loads such as a Blumlein. Its charging source
is a MOSFET based inductive adder with output
impedance of 100 ohms and a sufficiently high Thevenin
voltage of 12 kV. It is better suited to charging a 12

Figure 5. The Blumlein configuration pulse forming network.

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cable and overvolting the spark gap rather than driving a


6 Blumlein with twice the transmission delay of the
single line. A Blumlein presents four times the effective
capacitive load of a single ended transmission line to a
slowly rising charger. A fast charging time much less than
one microsecond is desirable for operating the spark gap
in the overvoltage mode for faster switching time. To
achieve overvoltage, a low impedance, lower voltage
charger is suited for Blumleins, while a high impedance,
high voltage charger is better suited for single ended
transmission lines. Slow chargers may be used in conjunction with a fast spark gap trigger source that delivers a
low current pulse to initiate the spark gap discharge.
The minipulser Blumlein pulse generator system was
designed specifically for cuvette loads and it has a compact flyback transformer charging source to charge the
Blumlein up to 5 kV in 1 s. Currently work is underway
to reduce the charging time of the Blumlein to less than
0.5 s. The Blumlein employs distilled water as a dielectric to reduce its overall size. Additionally, the liquid dielectric offers the freedom of easily placing the spark gap
within the waveguide plates.
The application of nsPEF during fluorescence microscopy is less demanding of voltage level, but more demanding of miniaturization, pulse flexibility, and variations in load impedance. The micropulser uses a single
power MOSFET, Directed Energy Inc. DE275-501N16A,
to directly switch the energy storage capacitor to the microscope cell chamber Figure 6.. Fast HV MOSFETs are
commercially available w4x with a rise time of -3 ns and
rated voltage of 1 kV. The charging source is a dc power
supply with a series-charging resistor to isolate it from
transient pulse current. The MOSFET direct drive topology was chosen for the microscope system for miniaturization and additionally because it offers extreme flexibility
of the pulse amplitude, width, and even rise time. The
sum of these features enables real-time imaging of cells
exposed to nsPEF with high flexibility in the experimental
variables of pulse shape.

PULSE GENERATORS FOR


CUVETTES

The medipulser single ended transmission line system


Figures 7 and 8. is flexible in pulse width repetition rate,

Figure 6. Solid state MOSFET pulse generator directly driving the


load.

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Vol. 10, No. 5; October 2003

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Figure 7. The medipulser sitting atop the chassis of its inductive


adder charger.

and has some flexibility in the amplitude. The charging


source for the transmission line is a MOSFET based inductive adder pulse generator. The inductive adder generates 150 ns pulses up to 12 kV and was designed for variable amplitude control. The amplitude of the medipulser
output to the cuvette is controlled by adjusting the charging voltage andror the spark gap electrode spacing. It is
not convenient to adjust amplitude because the spark gap
spacing largely sets the output voltage, but it is done within
the range of 1 to 4 kV. The pulse width is flexible because
the coaxial cable transmission line is interchangeable with
cables of other lengths. Each 12 cable bundle is terminated with medium high voltage MHV. interconnects
Figure 4. to allow other cables lengths to be swapped
into service. The pulse repetition frequency and exposure
duration are easily controlled by a logic level trigger oscillator and pulse counting gate, respectively, integrated with
the inductive adder charging source.
The minipulser Blumlein system w5x is more compact
Figures 9 and 10. and better suited to cuvette loads than

Figure 8. A 2.5 kV, 6 ns pulse from the medipulser. Vertical scale


is 1000=. Measured with 500 MHz resistive voltage divider mounted
to the cuvette chamber.

Figure 9. The minipulser system with charger top. and Blumlein


bot..

coaxial cable transmission line systems because it uses water as a dielectric. Waters high dielectric constant of 81
makes the Blumlein physically shorter in length for a pulse
of given length than pulse forming lines made from plastic
and most ceramics. Additionally, the use of water both for
the load and the pulse forming line makes impedance
matching a simple matter because the width and separation dimensions of the Blumlein waveguide conductors are
similar to the dimensions of the cuvette Figures 10 and
11.. The minipulser has some flexibility in amplitude by
varying the charging voltage andror the spark gap spacing.
The pulse width is fixed by the design of the Blumlein.
Repetition rate and exposure duration are easily controlled as with the medipulser.

PULSE GENERATORS FOR


MICROSCOPY

The micropulser pulse generator is suited for nsPEF exposure to cells on a microscope slide and for real time

Figure 10. A 3 kV, 7 ns pulse from the minipulser. Vertical scale is


1000=. Measured with 500 MHz resistive voltage divider.

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Behrend et al.: Pulse Generators for Pulsed Electric Field Exposure of Biological Cells and Tissues

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Figure 11. Physical model of the Blumlein. The center electrode is


the shield of both lines combined together.

microscope slide. The tradeoffs between each of the three


pulse generators have been discussed in terms of pulse
characteristics, flexibility, demands on the charging source,
and physical dimensions. A water Blumlein pulse generator is preferred for high electric field exposures to cuvettes because the charging circuit does not have to generate voltage equal to twice the desired load voltage as in
single line pulse forming networks. Even more advantageous is that the compact nature of the water Blumlein
with its similar dimensions to the cuvette yields a more
precise impedance match to the load by minimizing series
inductance. Direct drive solid state pulse generators can
offer the most flexibility in amplitude and pulse width,
and rise time, while being very compact. MOSFET direct
drive is suitable for nsPEF voltage requirements of less
than a kilovolt. Pulsed power miniaturization with solid
state devices enables integration of the generator with the
load and the fluorescence microscope for real time investigation of nsPEF applied to cells.

ACKNOWLEDGMENT
This work was supported by AFOSR and the ARO.

REFERENCES
Figure 12. A 400 V, 40 ns pulse to cells on the slide with the micropulser. Vertical scale is 1000=. Measured at the MOSFET drain
with a capacitive coupled voltage divider.

observation during pulse delivery w6x. The micropulser


provides very high flexibility and simple control of amplitude, pulse width, repetition rate, and duration. The output pulse Figure 12. replicates the TTL level trigger signal in width and repetition sequence. Additionally, the rise
time of the pulse can be controlled within a range of 3 ns
to 15 ns by adjustment of the MOSFET gate signal amplitude. The direct drive MOSFET micropulser offers the
most control of pulse parameters, but cannot match an
overvoltaged spark gap in pulse switching speed or high
voltage amplitude.
The micropulser is built onto a standard stage insert
Figure 2. for retaining slides on a fluorescence microscope stage. The micropulser holds the slide within its own
platform having two copper contacts to mate with the
platinum electrodes on the slide surface. The pulse generator occupies minimal space on the stage because it is built
onto a stage insert plate, and it accommodates real time
observation of the cells during nsPEF exposure.

w1x S. Beebe, P. Fox, and L. Rec, Nanosecond Pulsed Electric Field


nsPEF. Effects on Cells and Tissues: Apoptosis Induction and
Tumor Growth Inhibition, IEEE Trans. Plasma Science, Vol.
30, pp. 286292, 2002.
w2x M. Gundersen, P. T. Vernier, and L. Marcu, Ultrashort Pulse
Electroporation: Applications of High Pulsed Electric Fields to
Induced Caspase Activation of Human Lymphocytes, Proc.
IEEE Power Modulator Conf., p. 667670, 2002.
w3x S. Xiao, S. Katsuki, S. Kono, M. Moselhy, and K. H. Schoenbach, Recovery of Water Switches, Proc. IEEE Power Modulator Conference, p. 471, 2002.
w4x IXYS RF, 2401 Research Blvd., Suite 108, Fort Collins, CO
80526, USA.
w5x A. Kuthi, T. Vernier, and X. Gu, Compact Nanosecond Pulse
Generator for Cell Electroperturbation Experiments, Proc.
IEEE Power Modulator Conf., p. 225, 2002.
w6x M. Behrend, A. Kuthi, and P. T. Vernier, Micropulser for
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Power Modulator Conf., p. 358, 2002.

SUMMARY

WO transmission line based pulse generators have


been presented for exposing cells in cuvettes to nsPEF,
and one pulse generator for applying nsPEF to cells on a
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IEEE Transactions on Dielectrics and Electrical Insulation

Vol. 10, No. 5; October 2003

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r1r

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