Axon Axopatch 200B, Theory Of Operation

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USE OF THE PATCH CLAMP – A TUTORIAL • 29
Chapter 3
Set the external command for a 5 Hz, 100 mV
p-p
1 rectangular waveform. This will cause a
200 pA
p-p
2 current to be forced through the model cell. Switch the MODE to I-CLAMP
NORMAL. The signal at the SCALED OUTPUT is now V
m
. Set the output gain to x10.
There should be approximately 1 V
p-p
3
square waveform (V
m
≈
100 mV
p-p
) with a time
constant (
τ
) of 16.5 ms (± 15%, 10 - 90% rise time of 36.3 ms) that is identical to the
τ
of
the model cell.
To observe the two different speeds of the I-CLAMP loop and at the same time confirm
that the current clamp loop settles much faster than V
m
of the model cell, connect an
oscilloscope channel to the I OUTPUT BNC. Switch the rear panel slide switch near the
BNC to 100
β
mV/pA ("up" position).
The observed current signal will have an amplitude of 20 V
p-p
. With the MODE switch set
to I-CLAMP NORMAL the 10 - 90% rise time will be about 250 µs with about 10%
overshoot. Switch the MODE to I-CLAMP FAST and the response will change to a rise
time of about 35 µs with less than 10% overshoot (the internal rise time under these
conditions is about 10 µs, the signal at the I OUTPUT BNC is filtered at 10 kHz and thus is
limited to a rise time of 35 µs).
SERIES RESISTANCE compensation is functional in I-CLAMP mode when
CORRECTION is on, and may be used to compensate for the pipette resistance. This is
most important during intracellular or whole-cell measurements when one wishes to
measure the membrane potential or pass current to control the membrane potential. Since
the fraction of series resistance compensation applied is determined by the amount of
CORRECTION, set the CORRECTION knob to 100% when using series resistance
compensation in I-CLAMP mode. However, remember to reduce the amount of
CORRECTION before returning to V-CLAMPmode in order to avoid oscillations.
Viewing SCALED OUTPUT, use the SERIES RESISTANCE knob to bring the voltage
level during the pulse to the baseline level. Membrane changes may now be more
accurately recorded and injected current (using the HOLDING COMMAND controls) more
reliably used to control membrane potential.
1
mV
p-p
- mV peak-to-peak
2
pA
p-p
- pA peak-to-peak
3
V
p-p
- V peak-to-peak