When patching a cell, what exactly is the electrode doing that "injects" current into the cell? If there's no literal injection of charge/current into the cell, how can the cell do things like accumulate charge, progressively depolarize with constant current, etc. I'm looking for a very nuts and bolts explanation of how this works.
Thanks!
Have you sat down in a quiet place with the Axon Guide? It covers most of this stuff in a reasonably digestible way. Generations of patch clampers have used versions of it.Free download available from Molecular Devices:
http://mdc.custhelp.com/euf/assets/content/Axon%20Guide%203rd%20edition.pdf
If you want to understand this then start from the parent method - (straight) voltage clamp - as invented by KC Cole (he showed Hodgkin and Huxley how to do it on the squid giant axon and they used it to establish the ionic hypothesis of the action potential - and get a Nobel prize).
The object is to keep the voltage constant. To do this you need one electrode to measure the voltage and another electrode to pass current, positive or negative to stop the voltage from changing. Thus, the current you record is actually 'feedback' current. that's the real reason why cation currents are recorded in the negative direction - its the feedback current to stop the cell's inward cation currents from changing the membrane potential.
However, you don't see much if you hold the voltage constant so the voltage clamp permits you to suddently change the clamped voltage to a new level (usually depolarized but it can be either direction). This triggers voltage sensitive ion channels to open and admit ions - carrying charge. Now the feedback loop in the voltage clamp amplifier has to pass an equal and opposite current to clamp the voltage at the new set potential. Congrats you just did a step voltage change.
Really that's all there is to the principle. Another time I'll talk about capacitance because this is the property of the cell membrane that makes voltage clamp recording necessary.
Incidentally, I was in the Cole lab (after he left). I saved some of his equipment from being trashed (in Woods Hole). Recently I discovered how important that was as I have established that I have the original voltage clamp chamber that Cole (and Marmot) used for voltage and current clamp recordings on the squid giant axon. If anyone is interested I am happy to present this extraordinary piece of biophysics history (I already did to the Biophysical Society).
Patch clamping is voltage clamp but cleverly adapted so that you only need one electrode. However, beware. In actuality patch clamp really clamps the inside of the patch electrode, its an assumption that the attached cell will also be effectively clamped (generally valid for a round cell such as a DRG neuron but unlikely to be true for a highly ramified cell with dendrites).
I hope this is useful by all means drop me a line.
For a beginning eletrophysiologist and as a helpful reference for the more experienced, I strongly recommend Patch Clamping: An Introductory Guide to Patch Clamp Electrophysiology by Areles Molleman. It has fantastic diagrams and it cuts out a lot of jargon, while maintaining an appropriate level of scientific terminology. I recommend it to all my students, so I recommend it to you as well.
I've attached a PDF of one of the appendices of my PhD thesis on just this topic. It includes an analysis of a simplified voltage clamp circuit, as well as it's limitations.
Apart from what the others rightly mentioned, with respect to whole-cell voltage clamp I can only recommend you the following PDF by Boris Barbour (only 23 pages!):
http://www.biologie.ens.fr/~barbour/electronics_for_electrophysiologists.pdf
It not only shortly summarizes basic electronic principles of electrophysiology but also covers the most important capacitive artefacts, the errors that derive from that and the usually used compensation mechanisms.
Try read this link http://www.utdallas.edu/~tres/microelectrode/microelectrodes_ch02.pdf
may be it will helpful
Hello,
You could watch this video to get a broad idea.
https://www.youtube.com/watch?v=qHN_AjXIFPA
I am agree with George WJ Wright . This book is really amazing and has a very simple language: An Introductory Guide to Patch Clamp Electrophysiology by Areles Molleman. Although my background is Fluid Mechanics, I understand every single word of this book.
https://en.wikipedia.org/wiki/Electrophysiology
Unlike in the voltage clamp mode, where the membrane potential is held at a level determined by the experimenter, in "current clamp" mode the membrane potential is free to vary, and the amplifier records whatever voltage the cell generates on its own or as a result of stimulation.