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46 Compensation Procedures
7.3 Offset Compensation
In all patch clamp configurations a number of offsets have to be taken into account. These include amplifier offsets
(
±
30 mV), electrode potentials (
±
200 mV, depending on Cl
−
concentration of pipette and reference electrode),
liquid junction potentials, and potentials of membrane(s) in series with the membrane under study. Some of these
offsets are fixed during an experiment (like amplifier and electrode offsets), some are variable. It is standard
practice to take care of the fixed offsets by performing a reference measurement at the beginning of an experiment.
Thereby an adjustable amplifier offset is set for zero pipette current. Thereafter the command potential of the
amplifier (displayed as
V-membrane ) will be equal in magnitude to the membrane potential if no changes in offset
potentials occur. The polarity of the command potential will be that of the membrane for whole cell and outside out
configurations but will be inverted in the cell-attached and inside out configurations. In cell-attached configuration
an additional offset is present due to the resting potential of the cell under study. Liquid-junction potentials may
appear or disappear during the measurement when solution changes are performed or in the case that the pipette
solution is different from the bath solution (Barry & Lynch, 1991; Neher, 1992; Neher, 1995). Conventionally,
these problems are handled by applying the appropriate corrections and sign inversions during offline analysis. The
Patchmaster
software allows this to be done at the time of data acquisition, considering the relevant Recording
Mode , which may or may not result in inversion of holding and commanded voltages. In addition, it offers three
features which interact in a way to provide for simple online handling of offsets, such that V-membrane reads the
correct membrane potential (correct both in polarity and magnitude) at all times. These are:
V
0
: An adjustable hardware offset-voltage. The actual potential applied to the pipette is the sum of V-membrane
and V
0
.
Auto- V
0
: An automatic operation which systematically varies V
0
for zero pipette current. Before doing so, the
procedure sets V-membrane to an appropriate value (see below).
LJ: A software variable which can be set by the user. It represents the sum of all ”variable offsets” applicable at
a given time (Offset Sum). In order for the variable LJ to actually represent the various offsets, and to correct for
these, three conditions have to be met:
ˆ
The user has to calculate LJ correctly, according to the rules outlined below. This value should then be
entered in the LJ control.
ˆ
An Auto- V
0
operation has to be performed at the start of an experiment. The software will then set V-
membrane to -LJ (for Out Out and Whole Cell mode) or +LJ (for On Cell and In Out modes) at the start
of an Auto- V
0
operation.
ˆ
During experiments, V
0
has to be changed in parallel with user-induced changes in LJ. This is done auto-
matically by
Patchmaster
.
An analysis of the underlying offset problem and justification for the procedures can be found in Neher (1995).
The rule for calculating the Offset Sum (LJ) is to form the sum of all changes in offsets which occur between the
reference measurement and the test measurement. The polarity of a given offset voltage should be taken as viewed
from the amplifier input (positive, if positive side of the voltage source is closer to the input). A sign inversion has
to be applied if the offset under consideration disappears. A procedure how to measure liquid junction potentials
is described in Neher (1992). Ion mobility for calculation of liquid junction potentials can be found in Barry &
Lynch (1991). The table below lists the values for some typical solutions.
In each case, a liquid junction potential between the given solution and physiological saline (main salt: 140 mM
NaCl) is listed. Polarity is that of physiological saline with respect to the given solution (according to the convention
of Barry & Lynch).
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