off-set

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zang
zang's picture
off-set

Soory for stupid question.

Prior to whole cell patch, normally people do off-set, so the current trace is on the base line (zero). However, the trace is sometimes not stable (probably due to the AgCl coating condition). In such case, the trace gradually goes out from baseline.
If the whole cell was performed in such condition immediately after off-set was done, what can be expected to influence the data after 30 min?  

Thanks.

recordingfan
recordingfan's picture
I have a related question.

I have a related question. The offset is usually quite different between when the eletrode is first touching the solution and when the eletrode is right above the tissue. When is the right time to zero the electrode, immediately after it is immersed in perfusion or right before patching?

Many thanks!

engstar
engstar's picture
It is a basic thing that u

It is a basic thing that u should do the offset zeroing when the pipette has touched the bath solution but has not touch the cell. 

The offset will have changed when you do the real test after sealing. You can correct this change after the experiment or during the experiment, which should not be difficult. Pls see Pages 147-153 in Single Channel Recording by Neher etc, in which a good account of the correction methods has been given.
 
You should not do offset zeroing after making a good seal!  At that moment even there exists a great offset, the current baseline may still be zero. That is, the zero offset current u get would be produced by the great seal resistance rather than your adjustment of the offset. 

recordingfan
recordingfan's picture
I think I wasn't make the

I think I wasn't make the question clear for engstar:

What I mean is that I often see significant drift of baseline when I am lowering my electrode in the solution, before touching anything. For example, the baseline is different when my electrode is 5 um above the tissue or 4000 um above the tissue. In both case, the eletrode is still in solution, without touching the tissue/cells I am going to record. I think in this case, the electrode should always be zeroed as soon as it is lowered to touch the solution, which is more equivalent to the ground. Any comment on this is welcome.

engstar
engstar's picture
in the course of pipette

in the course of pipette going through the bath solution, there may be some current baseline drift, which is caused by the diffusion of components of the bath solution and the pipette solution. as long as the drift is not too much, that is not a big deal. otherwise u should check whether ur silver wires need chloridation, or whether there is liquid film crawling along the wire or the margin of the bath chamber, or whether there is sneaky leak of solution.