Understand whole cell capacitance and series resistance

3 posts / 0 new
Last post
KaraP
KaraP's picture
Understand whole cell capacitance and series resistance

 Hello.

I am new to patch clamping and wantd to ask about whole cell capacitance and series resistance.

One I break into my cell, I clamp and -60mV and input a pulse of -4mV. I then put on wholce cell capacitance to reduce the fast component of my transients (the slow components remain). Once I have done this I apply series resistance. I normally just put on a bit of both until the fast transient component is gone (is this correct?). I then finally put on compensation. I am using an axopatch 200B amplifier.

Q1 Why do I want to cancel the fast part of the transients? Is it beause it gives me a rough measurement of the cell size? Is there any other reason? Do some people cancel all of the transients? Do some people not?
Q2 How to I calculate how much compensation to put on? I have been told different things by different members of my lab. 

Any help would be greatly appreciated!!!

lazy
lazy's picture
 Q1 Asmy understanding, the

 Q1 Asmy understanding, the fast part of the transient is to cancel the electrode capacitance. When you record the fast current (such like Na current), you may have a problem without cancelling the rast component. Cell size will be speculated by cancelling the slow component.

Q2  I do it with 80% in gengeral. Ideally 100%, but you will have occilation. 

Timmer
Timmer's picture
Hi Kara,

Hi Kara,

First thing to say is that you need to apply the fast transient cancellation BEFORE you go whole cell (ie. while still in cell-attached mode). This is because it is for cancelling the capacitance caused by the pipette. Cancelling this will allow you to get decent readings from your test pulse in whole-cell mode (eg. series resitance calculation), and will also prevent clipping artefacts due to brief transients during voltage steps.

The slow capacitative transient is formed in whole-cell mode by the RC circuit consisting of the membrane capacitance and Rseries. The slow capacitative cancellation circuit is designed to cancel this, and to feed the Rseries compensation.

The Rseries compensation is used because the series RC circuit can affect the voltage clamp, leading to delays in voltage response. The compensation (this time it's active compensation in the cell circuit, not just cancellation in the recording) works by over-injecting current when the holding potential is changed in order to speed up the response. The compensation circuit takes estimates of the RC time constant calculated from your Cslow cancellation, and feeds it into the current injecting circuit.

A perfectly Rseries compensated cell would produce a perfect clamp, but in practice this is dangerous as the compensation circuitry is susceptible to "ringing" - ie. oscillatory positive feedback, which will tend to nuke your cell/destroy your seal. The usual advice is to aim for about 80% compensation, but in some cases you will have to accept lower. My only advice for your specific case is to try and see how large a compensation you can introduce without damaging the cell. Unfortunately I use HEKA not Axopatch so I can't help you with specific settings!

Hope some of this helps!

Tim