Wolfgang Frey
Forschungszentrum Karlsruhe, Institut für Hochleistungsimpuls- und Mikrowellentechnik,
D-76021 Karlsruhe, Germany
Two years
ago, an experiment for monitoring the membrane charging of biological cells on
the nanosecond time was built up in cooperation with the Center for
Bioelectrics (CBE) at Norfolk, VA. Currently, at the CBE the membrane charging
of mammalian cells is investigated. The main objective at IHM is to
characterize the membrane charging of plant tissue. Both groups use a new
voltage sensitive dye, ANNINE-6, for the determination of the temporal
development of the transmembrane voltage during field exposure.
In
principle, the cells under consideration are stained by the voltage sensitive
dye and exposed to an external electric field. At a certain time during field
exposure, the stained cells are illuminated by a 5 ns long laser pulse. The
response from the cells is monitored by a fluorescence microscope. The
intensity of the fluorescence response from the dye molecules attached to the
membrane gives information about the membrane voltage at the time of
illumination.
After an
introduction into the technique, the presentation shows first results of the
fluorescence response from stained onion tissue. 100 ns after the beginning of
the field exposure, all membranes of the cells of the onion tissue show a
depolarization, which is a discharging of the membrane beginning at the resting
potential (-50mV) to positive values.
This is in
contrast to the theoretical expectations. Membrane sections facing the Anode
should show a hyperpolarization, i.e. a charging of the membrane from the
resting potential to negative values.
To clarify
this discrepancy to theory, experiments on HEK293 cells were started.
Calibration curves for this cell line are known from literature. The results of
the fluorescence response from HEK293 will be discussed in detail for different
excitation wavelengths (440nm and 468nm) and for different values of the
external electrical field strength.