Axon Axopatch 200B, Theory Of Operation

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USE OF THE PATCH CLAMP – A TUTORIAL • 17
Chapter 3
the command potential of 40 mV amplitude and about 10 ms duration. If you are using
the SEAL TEST it will generate a 5 mV command instead of a 40 mV command. You
should scale the amplitude values in the following paragraphs accordingly.
6) Initially select a 10 kHz or 100 kHz bandwidth since this will allow the fast capacity
transient to be more readily distinguished from the slower whole-cell transient
(Figure 3a). At this band-width use the FAST MAG and FAST
τ
of the PIPETTE
CAPACITANCE COMPENSATION to eliminate the fast capacity transient as
previously described. The fast transient can be distinguished from the slower whole-
cell transient by its more rapid time course. At a sweep speed of 50 or 100 µs per
division and a vertical sensitivity of 0.5 or 1.0 V per division attempt to make the
leading edge of the whole-cell transient look similar to Figure 3b.
7) Reduce the bandwidth to 5 kHz. At a slow sweep speed ( e.g. , 5 ms/division) and high
vertical resolution ( e.g. , 20 mV/division) adjust the LEAK SUBTRACTION
potentiometer (see Adjustment of Leak Subtraction above) to eliminate leak current
from the 500 M
Ω
resistor simulating the membrane resistance in the model cell as in
Figure 3c.
8) Turn the WHOLE CELL CAP. switch on. Reduce the vertical resolution to 1 V/div
and increase the sweep speed to 1 ms/div. Using the WHOLE CELL CAP. and
SERIES RESISTANCE controls simultaneously, minimize the capacity transients as in
Figure 3d. There may be a small wiggle at the leading edge of the capacity transient
due to minor misadjustment of the fast electrode compensation controls which can be
removed by readjustment of those controls. Final adjustment should be done with a
vertical resolution of about 50 mV/div. (Note: Be sure that the trace is flat at times
beyond about 1 ms after its start. This is best accomplished with a sweep speed of 1 or
2 ms/div.) With a little practice it should be simple to reduce the capacity transient into
the noise. The values of the series resistance and whole-cell capacitance determined in
this way will be quite close to the correct values.