Guide to Electrophysiological Recording
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37
Chapter 4
headstage with your other hand so that the micromanipulator will not have to absorb
your force. Apply about 30 mbar of positive pressure to the holder tubing, then lower
the pipette tip into the bath. Any voltage offset between the bath electrode and the
patch electrode will show up as a non-zero tracking voltage on the I (nA) meter of the
MultiClamp Commander. Press the Pipette Offset button to null the offset. Remember
that the Pipette Offset does not permanently remove liquid junctional potentials in
whole-cell recordings (the liquid junctional potential returns after the whole-cell
configuration is achieved).
Note:
Check the stability of your bath (ground) and patch (recording) electrodes.
Drifting electrodes will cause a continual current drift off zero, indicating that
the electrodes probably need to be rechlorided.
Check the Seal Test checkbox and observe the “Scaled Output: Membrane Current” on
a scope; the trace should resemble the top trace in Figure 3.1. Note the electrode
resistance by checking the Resistance checkbox. Lower resistances (2-4 M
Ω
) are
preferred for whole-cell recording (to minimize series resistance), but if the resistance
is too low it can be difficult to obtain a gigaseal. Higher resistances (>5 M
Ω
) are
obviously necessary for sealing onto smaller cells or processes. Apart from these basic
rules, choice of the appropriate electrode resistance is largely a matter of experience
and experimental design.
The method of approaching the cell depends upon whether it is in a “clean”
environment (cell culture) or “dirty” environment (intact tissue). For a cell in culture,
you can maintain the positive air pressure at about 30 mbar. Lower the pipette until it
just touches the cell. As you press harder, causing dimpling of the surface of the cell,
you will see the electrode resistance increase, appearing as a decrease in the size of the
current pulse (Figure 3.1, three upper traces).
For a cell in a piece of tissue (
e.g.
a brain slice) you should increase the air pressure to
about 80-120 mbar
before
the electrode tip touches the surface of the tissue. This is to
help the electrode punch through the surface debris. Once inside the tissue, it may help
to reduce the pressure to 30-50 mbar, so you are not simply blowing cells away from
the tip of the electrode. If you are “blind” patch clamping in a slice, slowly advance
the electrode while looking for a
sudden
increase in resistance, indicating that you have
