24
•
USE OF THE PATCH CLAMP – A TUTORIAL
Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
bandwidth turning ON and OFF this switch does not noticeably change the noise riding on the
waveform.
Despite the fact that the membrane potential is now being changed extremely rapidly (about
30 µs) in response to a step command of potential, two important effects of series resistance (R
s
)
have
NOT
yet been eliminated. These are IR drops resulting from the flow of ionic currents (I)
in the cell membrane and the filtering effect of the series resistance and cell membrane
capacitance on measured membrane currents. An ionic current of 2 nA flowing across a series
resistance of 10 M
Ω
will produce a 20 mV error in the true membrane potential relative to the
command potential; this effect of series resistance is well known. It is far less well understood
that series resistance in conjunction with membrane capacitance will have the effect of filtering
the measured current with a one pole RC filter with a corner frequency given by 1/2
π
R
s
C
m
. For
R
s
= 20 M
Ω
and C
m
= 50 pF, this is only about 160 Hz.
The CORRECTION potentiometer is used to greatly reduce both of these errors. To set
CORRECTION properly, first set the LAG potentiometer at about 10 µs. Without changing any
of the settings previously established, advance the CORRECTION potentiometer gradually up to
about 95%. Observe the current at a vertical gain of about 100 mV/div and a sweep speed of
about 500 µs/div. As the CORRECTION percentage increases, a small transient will emerge at
the leading and trailing edges of the command step. At the same time the noise in the measured
current will increase. By the time the CORRECTION potentiometer setting has reached 95%, a
ringing transient with a peak-to-peak amplitude of roughly 400 pA will have emerged (Figure
6g). The transient should "ring" (i.e., oscillate) and will reverse polarity 2 or 3 times before
disappearing into the noise somewhat less than 1 ms (in 5 kHz bandwidth) after the beginning of
the command step.
This residual transient is easily eliminated. Usually all that is required is to slightly (about 1%)
increase the setting of the WHOLE CELL CAP. potentiometer setting to make the residual
transient disappear into the noise (Figure 6h). If this is not sufficient, very small readjustments
of the SERIES RESISTANCE control and FAST MAG and FAST
τ
may also be required.
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