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Tutorial
can still be successful. At least this appears to be so for central nervous system
tissue, if higher voltages are used.
Selecting the Stimulation Parameters
The polarity of the voltage pulse should match the polarity of the charge on the
molecules to be delivered to ensure that there is an electrical driving force to move
the molecules towards the cell. To keep the molecules from leaking from the
micropipette the offset or holding potential should be opposite to that of the
molecules to be electroporated. Even though the appropriate polarity can be
predicted, it should be tested empirically. For example, ‘anionic’ fluorescent
dextrans often move as cations following repeated single-cell electroporation
stimuli. For dyes, the best way to optimize the pulse protocol is by watching their
movement.
Voltage pulse widths from a few
µ
s to tens of ms form pores. These pores form in
a few microseconds and close more slowly. Thus, the pulse protocols to produce
electroporation are quite simple. For most applications, simple rectangular pulses
suffice. The pulses must result in total channel open time sufficient for an effective
concentration of molecules to enter the cell. Not surprisingly, several different
combinations of pulse widths, frequencies, and train durations may work equally
well. For further flexibility an external waveform generator can be used.
Pulse amplitude and duration influence both the size of pore formation and the
distance that molecules of different motility can move. The size of the transport
molecules is particularly relevant when selecting a pulse protocol since
electrophoretic mobility through biological tissue is highly size-dependent. Smaller
molecules like fluorescent dextrans are electroporated using single, low-voltage
pulses, while larger molecules like DNA require multiple pulses at higher voltage.
Even so, fluorescent dyes are useful for testing micropipettes to be used for
electroporation of DNA, since tips with a geometry successful for dye transfer also
work well for DNA. In fact, 250 kD dextrans require electroporation conditions
similar to those for DNA.
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
