Using the Patch Clamp EPC9 Manual 78
the current level. Additional suction sometimes lowers the access resistance, causing
the capacitive transients to become larger in amplitude but shorter. Low values of
the access (series) resistance are desirable and, when R
s
-compensation is in use, it is
important that the resistance be stable as well. A high level of Ca
2+
buffering
capacity in the pipette solution (e.g., with 10 mM EGTA) helps prevent spontaneous
increases in the access resistance due to partial resealing of the patch membrane.
Capacitive Transient Cancellation
If the fast capacitance cancellation was adjusted (as described above) before breaking
the patch, then all of the additional capacitance transient will be due to the cell
capacitance. Cancelling this transient using the C-slow and R-series controls will then
give estimates of the membrane capacitance and the series resistance. The easiest
way of cancellation is provided by the Auto C-slow function which may be activated
by selecting the Auto button or the Whole Cell mode (if included in the macro
function). In cases where the C-slow transient has a short time constant (100 µs or
smaller) , some improvement of the overall compensation may be achieved by
alternating cycles of Auto C-fast and Auto C-slow (if this doesn't work satisfactorily
you may fine-tune the compensation controls manually). After an iteration or two, it
should be possible to reduce the transient to only a few percent of its original
amplitude. However, if the cell has an unfavorable shape (for example, a long
cylindrical cell or one with long processes), the cell capacitance transient will not be a
single exponential, and the cancellation will not be as complete.
Series Resistance Compensation
Series resistance (R
s
) compensation is important when the membrane capacitance is
large or when the ionic currents are large enough to introduce voltage errors. To use
R
s
-compensation, you first adjust the transient-cancellation controls (including C-fast
and τ-fast if necessary) to provide the best cancellation. Then you select R
s
-comp by
turning up the %-comp control to provide the desired degree of compensation.
Note: The “R-series” control determines (along with the “%-comp” control) the amount of
positive feedback being applied for compensation. It should be adjusted with some care, since
too high a setting causes overcompensation (the EPC9 will think that R
s
is larger than it is);
this can cause oscillation and possible damage to the cell under observation.
How you should set the R
s
-compensation controls depends on the approximate
value of the uncompensated membrane-charging time constant τ
u
, which you can
calculate as the product of the C-slow and R-series settings (for example, suppose C-
slow is 20 pF and R-series is 10 MΩ; τ
u
is then 20 pF · 10 MΩ = 200 µs). If τ
u
is smaller
than about 500 µs, you should use the 2 µs setting of the R
s
-compensation switch to
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