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135᎐149
Abstract
High frequency current and voltage measurements were used to determine passive electrical properties, such as the
polarization effect at intact membrane interfaces and field-induced electropermeability changes in the cellular materials during
direct current pulses. The time sequence of the electropermeabilization at the levelc of the cell membrane showed a similarity to
the breakdown phenomena observed in cell systems Žpotato, apple and fish tissues, as well as plant cell suspension cultures . when
a single pulse with critical or supercritical field amplitude is applied. A slight membrane breakdown phenomenon occurred in the
first few microseconds after the initiation of the pulse at a critical electric field strength of 150᎐200 Vrcm. Significant membrane
breakdown was observed when the field strength of the electric pulses applied directly on the cell systems was in the range of
400᎐800 Vrcm. At various field intensities, the electrical potential across a cell membrane reached a critical value of
approximately 0.7᎐2.2 V. The initiation of conductive channels across the membrane occurred within nanoseconds during the
charging process of the membrane, whereas the formation of a high-conductance membrane due to pore expansion took place
within a few microseconds. The application of a single pulse, even with supercritical field amplitude, does not necessarily cause an
irreversible membrane rupture. The insulating properties of the cell membrane can be completely recovered within several
seconds after the termination of the pulse. The biological and engineering aspects of the membrane permeabilization are
discussed in this paper. These data are utilized as the basis for the design and optimization of high-intensity pulsed electric field
applications in the areas of food science and biotechnology. 䊚 2000 Elsevier Science Ltd. All rights reserved.
Industrial rele¨ ance: This paper demonstrates the complexity of the membrane permeabilization effects of high intensity electric field pulses and
the potential to convert this basic knowledge into effective processes where controlled membrane permeabilization is required. It is further
encouraging to note how quantifiable the kinetics of reversible or irreversible membrane permeabilization can be identified.
1466-8564r00r$ - see front matter 䊚 2000 Elsevier Science Ltd. All rights reserved.
PII: S 1 4 6 6 - 8 5 6 4 Ž 0 0 . 0 0 0 1 0 - 2
136 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
ratio of the cells in the suspension was approximately insulator separating two conducting aqueous phases,
0.7. i.e. as a dielectric in a parallel plate capacitor ŽSchwan,
All samples were prepared for treatment as cylinders 1957, 1977; Pauly, 1964.. The application of an external
Žthe dimensions of the samples perpendicular to the electric field induced the charging process at the mem-
electrical field: 2 cm2 ; length: 1 cm.. brane interfaces. The relaxation time constant, , is a
basic macroscopic characteristic for cell membrane
2.2. Electric field pulses protocol charge, which depends on cellular size, membrane ca-
pacitance, and the conductivity of cell and extracellular
electrolyte. Therefore, a model for examining systems
The exponential electric field pulses were applied
containing cells which is sufficiently described by the
with a high intensity pulsed electric field apparatus,
relaxation time , should also reflect other electric
designed and constructed in our department. The cen-
properties of a cell.
tral components of the pulse generator consisted of a
Theoretical predictions of for a spherical cell model
capacitor charging unit ŽFUG, HCK 800M-20.000,
and for a planar bilayer membrane cell model Žcubic or
Rosenheim, D. with pre-selectable charging voltage
multilayer cell model. are shown in Table 1. The use of
and current, a 2-F storage capacitor ŽMaxwell,
噛30560, San Diego, CA, USA., a treatment chamber a cubic cell model for the analysis of electrical proper-
and a high voltage switch ŽBehlke, HTS 101-80-FI, ties assumes that the membrane regions involved are
FrankfurtrMain, D.. The pulse shape was monitored two planar areas on opposite sides of a cell. Consider-
on-line with a 100-MHz digital storage oscilloscope ing only the cell membrane geometry in the field direc-
ŽTektronix, TDS 220, Wilsonville, OR, USA.. The volt- tion is permissible for large cells in biological tissue
age and current at the treatment chamber were regis- which have a very small membrane-thicknessrcell-size
tered with a 75-MHz high voltage probe ŽTektronix, ratio Ž5 nm:50᎐100 m. ŽHayden, Moyse, Calder,
P6015A, Wilsonville, OR, USA. and a 100-MHz cur- Crawford & Fensom, 1969; Zhang & Willison, 1991;
rent probe ŽTektronix, A6312, Wilsonville, OR, USA. Kuang & Nelson, 1998; Angersbach, Heinz & Knorr,
connected to an amplifier system probe ŽTektronix, AM 1999.. The application of the cubic cell model accu-
503B, Wilsonville, OR, USA.. The rise time of the rately describes a simulated behavior during the appli-
chamber voltage was approximately 200 ns. cation of electric field in the case of a yeast cell Ž S.
The treatment chamber was equipped with two cere¨ isiae. with a linear dimension of 8 m ŽWeaver &
polished stainless steel cylindrical electrodes which were Barnett, 1992..
placed in the sample containing a PVC tube ŽHeinz, The comparison between theoretical and experimen-
Phillips, Zenker & Knorr, 1999.. The electrode area tally obtained charging time constants in Table 1 shows
was 2 cm2 . The gap was adjusted to 1 cm. The ohmic that the cell model with planar membrane areas pro-
resistance of the treatment chamber containing the cell vides a better description of the passive electrical be-
material varied in the order of 90᎐2500 ⍀ Žthe resis- havior of an individual cell, and, therefore, gives a
tance of the electrical leads was ; 1 ⍀ . in accordance more realistic basis for describing the charging process
with the degree of cell membrane permeabilization. and the breakdown of cell membranes during applica-
The peak field strength E0 was gradually increased tion of electric field pulses.
from 50 up to 2000 Vrcm, whereas the pulse duration In the following, the simplified cubic cell model with
was adjustable in a range between 0.9 and 25 ms. planar geometry is used for examination of cellular
The temperature increase in the treated samples due samples.
to Joule heating was low in all experiments. A single
pulse with a maximum field strength of 2 kVrcm
2.3.2. Discharging pulse and electrical properties of cells
caused a temperature increase of ⌬T s 0.55⬚C in the
system
sample Ženergy input 2 kJrkg.. Treatment with ten
A typical time course of the applied discharge pulse
repeated pulses Žfield strength amplitude E0 s 700
on tissues or suspensions with a high cell volume ratio
Vrcm. caused a temperature increase of ⌬T s 0.68⬚C
is presented in the Fig. 1. The relationship between
in the sample Žtotal energy input 2.45 kJrkg.. Hence,
current density, j, and field strength, E, gave the in-
temperature related alterations in conductivity were
stantaneous sample conductivity as s jrE. For the
not considered. The initial sample temperature was
calculation of the electrical properties of the cells, it
approximately 20⬚C in all experiments.
was assumed that the external electric field E0 in the
initial 8 s after the start of the pulse Žouter rise time
2.3. Theoretical considerations period, approx. 0.2 s. was constant.
A typical current flow across the cells in tissues or
2.3.1. Charging time constant and cell model suspensions within a short Žs range. period after the
The intact cell membrane can be regarded as an application of an external electrical field Žassuming the
138
A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
Table 1
Electrophysical properties of intact cell systems a
Sample Average Cell Relaxation time constant of the cell membrane Žs. calculated according to: Relaxation time
cell size electrolyte Spherical model Multilayer model Cubic cell constant
d c Žm. b conductivity ŽSchwan, 1957, 1977. c Žexperimental. e
ŽKuang & Nelson, 1998. modeld
c ŽmSrcm. dc 0.5⭈ m ⭈ d c q c ⭈ l m d c ⭈ Cm Žs.
1 1
s ⭈ Cm ⭈ Ž q . s 0 s
2 a 2 ⭈ c c l m 2 ⭈ c
Fig. 1. Current density, j, field strength, E, and effective tissue conductivity, , vs. time during a single discharge pulse of a 2-F storage
capacitor at various field strength amplitudes, E0 , through potato tissue. For E0 s 66 Vrcm, the peak current density was 0.27 Arcm2 and the
total pulse width was 15 ms.
Fig. 2. Time course of field strength Ža, b. and current density Ža⬘, b⬘. in a potato cylinder in the first 8 s of a pulse with a field strength
amplitude of 66 Vrcm Ža, a⬘. or of 880 Vrcm Žb, b⬘.. The complete pulse course is shown in Fig. 1. The Roman numerals in Ža⬘. are defined
within the text ŽSection 2.3..
140 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
passive electrical properties do not change. is shown in The following equation is related to first order differ-
Fig. 2a. As for a heterogeneous dielectric, the current ential equations which describe the interaction of sev-
time course could be divided into three characteristic eral membrane properties, i.e. the parallel combination
periods. In the first instant, during the rise of the of a capacitance Cm and a resistance Rm in the equiva-
electric field, electrical polarization and charging of the lent circuit with cell current ic s jc. Am .
‘geometrical’ capacity caused the rapid development of
current peak jmax Žcurrent jump, period I.. During the d
second period ŽII., polarization effects arose from the i c s i p s Cm if t - tpore Ž3.
dt
induced charge accumulation at the interface of intact
membranes ŽMaxwell᎐Wagner effect.. The third period For t - tpore the polarization current charges the
related to a time range of 3᎐5 . The polarization intact membrane. At t ) tpore , the formation of aqueous
process was terminated and the electrical current den- pores on the membrane interface occurred and the
sity decreased to a minimal value, jmin . When E0 s instantaneous current, im , flowing across the membrane
constant, jmin remained constant Žperiod III. and was developed. This depended on the fraction of the aque-
adjusted through the electrical conductance of the ex- ous pores and on decreased transmembrane voltages.
tracellular liquid. The cell current was given as:
The same effects for biological matter were postu-
lated in the frequency-domain as ␣- and -dispersion
ic s i p q im if t ) t pore Ž4.
ŽSchwan, 1957, 1977.. The Laplace transformation may
be applied to translate the Maxwell᎐Wagner effect at
the membrane interface in the DC field Žtime-domain. For an intact cell, the current ic was equal to the
to an AC field Žfrequency-domain.. The analysis of polarization current ip as a result of the charging of the
conductance spectra within the -dispersion range of insulated membrane through the cell electrolyte Žin
3᎐50 MHz ŽAngersbach et al., 1999. and of the conduc- case of high cell concentration . from the pulse genera-
tivity relaxation in the time range from 0.2 to 8 s after tor. With this assumption, Eq. Ž3. could be transformed
the beginning of the pulse Žsee Fig. 4, curve number 1. to give the current density, and could be written as
provided identical information for all materials ex- follows:
amined according to the passive electrophysical proper-
ties of the cell system, such as volume ratio of intact jc s jp s c ⭈ E0 ⭈ exp Ž ytr . Ž5.
cells in the sample, charging time constant of the cells,
or maximum and minimum values of sample conductiv-
where t is the process time and c is the specific
ity Ž max , min ..
electrical conductivity of the cell electrolyte. From Eqs.
The time dependent density of the direct current, j, Ž2. and Ž5. the transmembrane potential for an intact
flowing through the elementary layer Žorientated per-
membrane can be given as:
pendicular to the electric field. of the system contain-
ing cells after the application of an external electric
d
field E0 could be described as: s E w 1 y exp Ž ytr .x Ž6.
2 0
j s x⭈ jc q Ž 1 y x . ⭈ ja Ž1.
Eq. Ž1. shows the relationship between the instanta-
neous current density in the interior of the cells, jc ,
where x is the volume ratio of intact cells; jc is the and the total current density in the sample, j, which
density of current flowing in an average cell; and ja is could be measured. The values of x and ja in Eq. Ž1.
the current density, flowing through an elementary could be eliminated by analyzing the relationships given
non-cellular unit. by the exposure to a subcritical electrical field. For cells
Here, it was assumed that the layer thickness was with intact membranes at the onset and at the end of
equal to the average cell size and that the cells were the polarization process waccording to Eq. Ž5.x the fol-
uniformly distributed within the sample. lowing equations applied:
The field-induced time-dependent voltage drop across
a cell membrane Žneglecting the natural potential of a
At t ª 0 jmax s x⭈ c ⭈ E0 q Ž 1 y x . ⭈ j a Ž 7a .
biological membrane, which was in the order of 10y2 V
ŽTsien, Hess, McClesky & Rosenberg, 1987; Tsong,
1996. can be expressed by Eq. Ž2.: At t G 5 jmin s Ž 1 y x . ⭈ ja Ž 7b .
tic time periods t ª 0 and t G 5 , respectively Žsee Fig. where lm is the membrane thickness Ž; 5 nm.. Eq. Ž11.
2aX .. Hence, Eqs. Ž1., Ž7a. and Ž7b. give: is valid when the resistance of the lipid area is very
high and the solution conductivity in the individual
jmax y jmin pore is unchanged.
js ⭈ jc q jmin Ž8.
c ⭈ E0
Eq. Ž8. allowed the calculation of the current jc 3. Results and discussion
flowing in an average cell interior from one experimen-
tal current᎐time curve when the membrane is intact. In the present study, the pulse width of fully expo-
During the application of high field strength pulses, nential capacitor discharges through the material ex-
membrane rupture occurs, and the cell current jc can amined was strongly dependent on the effective sample
be determined as follows. Assuming that the contribu- resistancerconductivity, which varied in correspon-
tion of the current density jmin , flowing through the dence to with the applied field strength. In Fig. 1, this
extracellular part, to the total current density of the is illustrated using potato tissue.
cell system with intact or permeabilized membranes Obviously, in the experiment with a pulse amplitude
remained unchanged, the cell current density jc in the of E0 s 66 Vrcm, the field strength was not sufficiently
case of a ruptured membrane could be described as: high to initiate electropermeabilization of the cell
membranes. The discharge current only passed through
c ⭈ E0 ⭈ w j y j⬘min x the extracellular matrix of the potato tissue because
jc s Ž9.
jmax y j⬘min the intracellular electrolyte was isolated by intact mem-
branes. The effective conductivity remained at its lowest
with value wapprox. 0.33 mSrcm, which is common for the
measurement of passive electrical properties of intact
jmin ⭈ E0 potato tissue with DC or AC in the 1᎐5 kHz range
j⬘min s Ž 10.
Esub ŽShaw & Galvin, 1949; Hayden et al., 1969; Angersbach
et al., 1999.x. Consequently, the current density and
where jmin is obtained from experimental data by the field strength decayed exponentially, with the highest
application of a first pulse on a subcritical field strength time constant of ; 3 ms.
level, Esub Žfor examined cell materials, as a rule lower In contrast to the first test for the pulse application
than 100 Vrcm.. The values of jmax and j were ob- with an amplitude of 880 Vrcm, the field strength E
tained from recorded current᎐time curves by the appli- decayed exponentially with a very low time constant,
cation of a second pulse with critical or supercritical approximately 200 s. The total sample conductivity
field strength Ec in the same sample. increased to 5.5 mSrcm as a result of the elec-
For the ruptured membrane, the relationship tropermeablization of previously insulating cell mem-
between a membrane current, im , and the transmem- branes.
brane voltage wEq. Ž2.x gave the membrane conduc- Fig. 2 illustrates the detailed time course of j and E
tance, Gm s im r, which could be attributed to the sum in potato tissue in the first 8 s after the initiation of
of the instantaneous conductances of all individual the pulse with amplitudes of 66 and 880 Vrcm Žthe
transient pores. The determination of the membrane complete pulse sequence is shown in Fig. 1.. Through
current, im , deduced from Eq. Ž4., was based on the the application of a sub-critical field strength, the exis-
assumption that the membrane capacitance, Cm , tence of insulated membrane properties was clearly
changed only negligibly during the formation and detected by means of an exponentially decaying polar-
widening of the pores. This assumption holds because ization current in the cells within the first few mi-
only a small fraction of the membrane was occupied by croseconds. With a field strength level of E0 s 880
aqueous pores ŽFreeman, Wang & Weaver, 1994; Win- Vrcm, the current through the samples showed a devi-
terhalter, Klotz & Benz, 1996., and the polarization ation from exponential decay at 0.4 s, indicating that
current ip exponentially decays even further after mem- electric breakdown in the cell membranes had oc-
brane breakdown. curred.
Assuming that the increase in membrane conductiv-
ity during field-induced breakdown resulted from the 3.1. Membrane polarization and electroporation
formation of cylindrical pores with ohmic behavior, the
time-dependent total Žaqueous. pore area, Aw , of the The changed conductivity of the total sample in the
membrane with cell liquid-filled pores was given by: pulse experiment yielded integral information about
the extent of the pulse-induced changes in the struc-
lm ⭈ Gm tural properties of the complete cell system. The time
Aw s Ž 11.
c sequence and dynamics of the electropermeabilization
142 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
Fig. 4. The field induced permeabilization in the first 12 s after pulse start with various field strength amplitude E0 ŽVrcm. for: potato tissue
Ža.: 1᎐100, 2᎐250, 3᎐400, 4᎐680, 5᎐1000, 6᎐1700; apple tissue Žb.: 1᎐100, 2᎐270, 3᎐550, 4᎐750, 5᎐1100, 6᎐1700; culture cell suspension Žc.:
1᎐110, 2᎐250, 3᎐330, 4᎐590, 5᎐1100, 6᎐1800; fish tissue Žd.: 1᎐80, 2᎐300, 3᎐500, 4᎐900. The effective conductivity course was calculated from
monitored current and voltage variation with time. The shortest total pulse width in all experiment was 900 s.
sample show a deviation from exponential decay in the speed of pore formation suggests that this action was
short time scale after pulse initiation. This indicated located at the lipid domain of the cell membrane
that electric breakdown in the membranes of individual rather than at protein channels. Pore initiation was
cells occurred during the charging process, i.e. in the most likely triggered by defects in the lipid structure
time period of 3᎐5 . In all experiments, the charging and occurred in the range of nanoseconds ŽCoster &
process was followed by a rapid development of the Zimmermann, 1975; Benz, Beckers & Zimmermann,
membrane current which was caused by a rupture and 1979; Gruenwald, Frisch & Holzwarth, 1981; Zimmer-
the evolution of high conductance membranes, initi- mann, 1996.. The pore widening of the lipid bilayer
ated by the critical or supercritical electrical field. happened in the microsecond range ŽSerpersu, Kinosita
With increasing field strength, the charging time up & Tsong, 1985; Ho & Mittal, 1996.. Other issues also
to the moment of membrane rupture was reduced, and needed to be considered. If a protein channel was
the rate of pore widening and the total area of the punctured by an electrical potential, the local heating
pores of the membrane increased. This was reflected by due to excessive membrane current might thermally
the rise in conductivity of the samples after apparent denature the protein Žor modify it electrically ., a reac-
membrane permeabilization ŽFig. 4.. tion known to occur in the range of milliseconds᎐sec-
onds ŽKim & Baldwin, 1990; Tsong, 1996.. Within a cell
3.2. Biological aspects of the membrane permeabilization membrane, the opening and closing of protein channels
could be controlled by the transmembrane electrical
Several hypotheses have been established that rea- potential. The gating potential of a typical ion channel
son that in real cell membranes, pore initiation may is in the range of 50 mV ŽTsien et al., 1987; Tsong,
occur in lipid domains or at protein channels 1996.. The argument that an intense pulse may force
ŽChernomordik et al., 1987; Glaser, Leikin, Cher- these channels to open before the breakdown of the
nomordik, Pastushenko & Sokirko, 1988; Tsong, 1990; lipid bilayer occurs may not be valid, because the
Zimmermann, 1996; Ho & Mittal, 1996.. In all our charging curve up to the breakdown point Žand also by
experiments it was observed that the immediate initia- super-critical field intensity . showed an exact exponen-
tion of conductive channels across the membrane and tial course with a time constant , which was character-
the rapid formation of a high conductance membrane istic of an intact unchanged membrane Žsee Figs. 2 and
due to their expansion took place in less than 1 s. The 4.. The simultaneous opening of protein channels and
144 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
the pulse. Under these conditions, pulse parameters branes. Electrical breakdown experiments on cell mem-
such as magnitude, the shape of the decaying electric branes have also led to the conclusion that local heat-
field, and the total pulse duration determined only the ing may play an important role after the primary process
secondary pulse effects, such as lifetime of the pores of electrical breakdown ŽCoster & Zimmermann, 1975;
already formed. Benz et al., 1979..
Once a membrane breakdown occurs, a current, im , The high conductance of the membrane during the
flows through the aqueous pore. Joule energy is gener- voltage relaxation following a pulse with supercritical
ated during the field application. An integration of field strength amplitude remained unchanged even
i2m rGm over the full pulse duration wassuming that at 8 when the current and voltage started to decay by the
s, the value of Gm is approx. 5 = 10y3 S Žsee Fig. 3c., experiment with E0 s 880 Vrcm ŽFig. 1.. With this
and that further conductance of the perforated mem- amplitude, the sample conductivity during the total
brane changes during the pulse proportionally with pulse width did not change significantly. However, it
decreasing sample conductivityx showed that for the showed a tendency towards the recovery of the initial
pulse experiment with E0 s 880 Vrcm presented in low conductivity state. In experiments with an ampli-
Fig. 1, the transmembrane electric field energy gener- tude of 440 Vrcm, the significant decay of the sample
ated during a pulse of 1 ms duration was approximately conductivity began only when the external field was at
2.5= 10y9 J. The dissipated energy in the small aque- half of this level ŽFig. 1.. Furthermore, the decay of the
ous zones of the porated membrane Žat approx. 8 s sample conductivity to its initial state Žthe conductivity
after the start of a pulse, considering a total pore area course extrapolated to the end of the pulse was approx.
of 46 = 10y1 2 m2 and a membrane thickness of 5 nm, 0.3 mSrcm. indicated that the membrane rapidly re-
yields a volume of approx. 2.3= 10y1 9 m3 . resulted in sealed during the voltage relaxation. Also, in experi-
an enormous specific energy input, of the order of ments where the maximum changes of the membrane
MJrkg. In comparison, the energy input into potato conductance were reached Žan experiment of this type
samples was approximately 0.5 kJrkg and the resulting is shown in Fig. 1, by Eo s 880 Vrcm., the measure-
temperature increase was 0.14⬚C. Obviously, the enor- ment of the passive electrical properties Žstarted 2 s
mous energy concentration on the pores zone could after pulse termination. indicated that the sample con-
produce local thermal fluctuations and some secondary ductivity was identical to the measurement before the
effects on the elastic modulus of the biological mem- pulse was applied Ži.e. the insulation properties of cell
membrane were completely recovered.. A reversible
breakdown of the cell membranes was observed. This
phenomenon is established experimentally for a wide
range of biological cells and is used as a basis for the
electromanipulation of cells in the fields of medicine
and biotechnology ŽChang et al., 1992; Zimmermann &
Neil, 1996; Lynch & Davey, 1996; Ho & Mittal, 1996..
Monitoring of the current during the first 8 s after
starting the repeated supercritical pulse applications
resulted in reversibility after the first pulse and irre-
versible cell membrane rupture after several pulses
ŽFig. 7.. At repeated pulses, with intervals of 1 s between
them, the oscillographic tracking of the charging cur-
rent of the cell membranes for the first and the second
pulse were reproduced with high accuracy. This meant
that the structural properties of the membrane after
the first pulse were identical to those of an intact
membrane. This finding is essential for the appropriate
use of pulsed electric fields. Only if the membrane
structure is restored, does the induction of critical
membrane voltage and membrane breakdown occur
again. Also, for the third pulse, the current course
showed only a slight deviation from the exponential
decaying process. This meant that the membrane struc-
Fig. 6. Variation of Ža. field strength, E, Žb. current density, j, and ture was restored to a greater extent within 1 s after
Žc. effective tissue conductivity, , with time for potato tissue during
first 80 s after pulse start with pulse amplitude 440 Vrcm Ž1. and
the second pulse. The membrane current was observed
880 Vrcm Ž2.. Complete pulse sequence is shown in Fig. 1. Effective for pulse number 5 in addition to the polarization
tissue conductivity is obtained as s jrE. current at the start of the pulse. At 0.5 s, a slight
146 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
directly in the tissue. A field intensity with an ampli- applied, the membrane rupture occurred within 1
tude of more than 1 kVrcm was induced directly in the s after pulse initiation. At a very high field
cell material. strength, the membrane breakdown should occur
The tests in mixed systems showed that when the during the voltage rise time. For all of the observed
supercritical field intensity is applied, the specific en- electroporation behaviors, it was found that the
ergy input into particulate tissues per pulse can be only attainment of critical transmembrane poten-
calculated as: tial did not explain all essential aspects of electro-
poration. By the application of a pulse with super-
max T 2 critical field strength, the membrane was substan-
Qs H0 E Ž t . dt Ž 12 .
tially charged to higher voltages as compared to
critical field strength. This clearly emphasized the
where is the tissue density; EŽ t . is the effective field need to consider the dynamics of the membrane
strength in the particulate as a function of time Žmea- voltage build up.
sured or predicted from electric field distribution in the 2. The formation of a conductive membrane due to
treatment chamber with regards to high conductivity the widening of pores was not an instantaneous
state of the cell material.; T is the total pulse width; process and was strongly dependent on the field
and max is the maximum tissue conductivity. This strength amplitude, if the pulse duration was a few
consideration has been confirmed with suspended cell times more than . The larger the field strength,
agglomerates and individual cells. the more rapid the dynamics of the pore formation.
The maximum conductivity value, max , could also be The initiation of conductive channels across the
determined experimentally prior to the supercritical membrane occurred in nanoseconds, while the for-
pulse application via the measurement of passive elec- mation of high conductance membranes due to
trical properties by a DC pulse experiment with subcrit- their expansion took place in a few microseconds.
ical field strength Žas represented in Fig. 4, max s A rapid formation of pores in a lipid domain of the
jmaxrE0 . or by a high frequency AC field yielding the cell membrane might be possible. Although a dras-
so-called -dispersion. For plant or animal tissue, and tic change in the membrane conductivity during the
for large plant cells in suspension, the electrical pulsation occurred Žin the order of up to 105 ., the
properties of intact or totally ruptured membranes are fractional aqueous area of the membrane was found
identical within the characteristic frequency range from to be small and never exceeded 1% of the mem-
5 to 50 MHz ŽAngersbach et al., 1999.. brane area in the field direction.
3. The originally low conductance state of the cell
material could be reached in all cell materials
4. Conclusion examined within a couple of seconds after termina-
tion of the pulse Ži.e. the insulating properties of
The time sequence of the electropermeabilization at cell membrane was completely recovered.. When
the cell membrane level showed similar breakdown critical or slightly lower amplitude pulses were used,
phenomena for different cell systems. When a single the original low conductance of the membrane
pulse with a critical or super-critical field amplitude could be reached, even during the pulse. The appli-
was applied, three different phases were typically in- cation of a single pulse with super-critical ampli-
volved: Ži. the development of a critical transmembrane tude caused the reversible breakdown of the cell
potential due to charge accumulation at the intact membranes. The lifetime of the high-conductance
membrane surface; Žii. the termination of the insulat- state of the membrane and the reversibility of the
ing properties of the membrane Žmembrane break- structural changes was found to be the secondary
down. resulting in development of a conductive mem- effect of electric field interactions with the cells
brane; and Žiii. the opening time of the pores and the and was also influenced by the energy concentra-
tendency towards complete recovery of the initial insu- tion on the pores zone of the membrane.
lating properties of the membrane.
These results are in accordance with the observa-
1. Classical membrane breakdown phenomena oc- tions reported for biological and artificial membranes
curred for large cells at field strength levels in the in classical electroporation phenomena.
range of 150᎐200 Vrcm. When the electrical po- From an engineering perspective, the electric field
tential over the cell membrane reached a critical interactions with real food cell systems can be summa-
value of approximately 0.7᎐2.2 V, it resulted in rized as follows:
membrane breakdown. The polarization time up to
membrane breakdown was influenced by the field 1. The field strength of electric pulses directly applied
strength. When supercritical electrical fields were to the materials with a cell size of 50᎐120 m must
148 A. Angersbach et al. r Inno¨ ati¨ e Food Science and Emerging Technology 1 (2000) 135᎐149
cal field pulses on plant membrane permeabilization. Trends in irreversible modification of membrane permeability by electric
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