In 1991 Erwin Neher and Bert Sakmann received a nobel prize for the invention of the “Patch Clamp Technique” – a truly extraordinary achievement that considerably broadened the field of “electrophysiology”. Neuroscience is near unthinkable without this technique these days and its use has been enhanced, refined and broadened within the last years.
Yet, this impressive achievement in science is – like most Nobel Prize -worthy inventions – so “out there” that not everyone has heard of it or can even imagine. And maybe if you have heard of it, it’s too complicated to really get what the hell is going on.
Fret not! This blog post is my succinct introduction to what on earth we neuroscientists are talking about when we go and “clamp” some cells.
The electrical brain
Cells, especially brain cells, have unique electrical properties, and if you know barely anything about our nerves, you at least know they’re communicating via electrical pulses. Or maybe, you even know that the nerves that move your arms or transmit the sensations of your limbs and organs to your brain work like electrical data-highways that come together in your spine and your brain. In any case, electrical potentials (that is “differences between two electrical charges”) are very important for the function of all membranes and, when altered, make a great tool for communication between cells.
The brain is a big accumulation of nerve cells and their nourishers and supporters (called glia cells) that weave a tight network that is constantly in action. When doctors stick electrodes to your head for an EEG (Electro-Encephalogram) – for example in a sleeping test – it means they can listen in the accumulating electrical currents in your brain. Similar things happen during an EKG where they monitor the electrical activity of your heart muscles. But let us stay focused on nerve cells for now.
What the doctors catch during an EEG is merely a radio signal coming from millions of interconnected stations. They can capture the over all change in the noise level, detect rhythms and such. It’s a bit like listening to the universe – there’s so much in the range of your detector, that you pick up a lot all at once. It’s absolutely impossible, from the outside and through your thick skull, to measure the signal of a few stations only, let alone one station or one person within a station.
What if you could, though? What if you could listen in on the electrical ups and downs of a one single nerve cell?
Well, that’s what Neher and Sakmann set out to do.
How to patch a cell
Imagine you had either a piece of brain or a spread out network of nerve cells under a microscope (both is possible). In both cases, just like in the brain, they’d be embedded in a fluid that nourishes them, keeps them warm or at least provides protection from drying out. Nerve cells come in all sorts of shapes and slightly different sizes, but they have one thing in common: They are tiny. Touching a single cell with forceps or even a hair is impossible – both would be way too big. But if you really want to listen to a single cell, you must get really, really, really close.
However, since cells actually don’t send out signals by waves or by radio, but by very tiny changes in their electrical properties, you need to get really close to “listen in”. In fact, there have been approaches where wires have simply been stabbed into cells to force electrical changes – but there’s a gentler method – and that’s what Neher and Sakmann came up with.
If you take a small glass capillary and heat it, you can pull it and make it longer and longer until it breaks into two, leaving a clean tip. If done properly, the duct within the capillary will still be open on both ends – but the end where it broke will be a fine, open tip. This is the kind of tool that is so small, you can actually poke a cell with it. It’s called a “patch pipette”.
However, glass is very bad at conducting electrical signals and you now need to find a way to actually get a sensor for electricity near your cell. A second problem is size. Getting a patch pipette’s tip of a few micrometers width (that’s less than 0.01 mm) onto a spot that is only slightly bigger (usually less than 0.25 mm) is infinitely worse than threading a needle. It can’t be done by hand. You’d quiver and shake way too much and if you quietly want to record the electrical activity of a cell over minutes or hours, standing there holding a capillary is not going to do.
Let’s treat that problem first, right? You need a very fine mechanical arm to calmly and without twitching approach the cell under your microscope. Way back when this was done with carefully constructed sets of screws that, when turned, inched the patch pipette forward until you could reach the cell. You have to imagine, though, that even when you touch your construction of screws, you’ll cause an earthquake when seen from the point of view of the cell or patch pipette. In our lab, we have only one setup left with such a mechanical manipulator. To cross the very last micrometers towards the cell so as to not stab it, a small crystal is behind the capillary. Upon changing an electrical field that surrounds the crystal, we can force this crystal to expand by only a few micrometers. This expansion is used for that final step towards the cell. This is just to show you how fine the movements must be. Luckily, today the so-called “micromanipulators” are comfortably moved by electrical motors that are so precise, that there’s little chance you will stab right through a cell or end up next to it, instead of on top of it.
What about the second problem, though? Glass is not a cable. You need at least a piece of wire to conduct any current. And that’s exactly what you’re gonna use. Remember that the cells we watch are usually swimming in some sort of liquid (much like in your body)? And do you also remember what you were told happens when you drop your running into the bathtub?
Good. Because you can use that knowledge.
Instead of stabbing the cell with your wire, you fill the patch pipette with liquid in which you dip a wire. Through the ions in the liquid (the composition depends a lot on what you wish to do with the cell) electricity can be conducted between the wire and the end of the capillary (and beyond). For example, once you’ve set down your capillary on the cell’s membrane, you can tune in on the electrical activity of that membrane. The wire is usually covered in Silver Chloride (AgCl) for a good conductance.
This, in theory, sounds pretty complicated, but quite possible since you’ve got fine tools for everything these days. Don’t underestimate that even patch pipettes have to be “pulled” by machines and that cells are very sensitive to the pH-values and osmolarity (that is, the number of particles per liter) that surrounds them.
Of course that picture is nevertheless extremely simplified. For example, cells aren’t always clean and neither is the liquid they’re swimming in. It wouldn’t matter much to have a few pieces of dust getting caught in your bath, but can you imagine how easy it is to get an opening of not even 2 micrometers (again, 0.002mm) clogged? Dust works just fine. What “electrophysiologists” do to avoid that is connect a little tube to the patch pipette and carefully blow air into it. Often, this is actually done with the mouth, but it can also be applied through a syringe. The pressure and small outflow of liquid from the capillary will avoid the clogging.
There’s another advantage to the mouth-to-patch-pipette connection: Once you have your capillary on the cell, you can carefully suck instead of blow. Try that with a nice bendable (or non-bendable) straw on your hand. The skin will be sucked into the straw and the edges of the straw “sealed” to your hand. This is, in principle what happens when you “suck” on a cell. You shut out the bath’s liquid out until it’s only you and your ‘patch’ of outter membrane under the straw… er… capillary. In electrical terms this seal will be so tight that the electrical resistance will exceed 1 GigaOhm [1,000,000,000 Ohm]. By comparison, the patch-pipette without the membrane usually has a resistance of maximally 10 MegaOhm.
Most researchers don’t want to look at a cell from the outside, though. They want to have access to the inside. This is also where we have to leave the analogy with the straw on your hand. Because the more you suck on that straw on your hand, the tighter the connection between your skin and your straw will get. It might even hurt. What your skin luckily won’t do, but cells will, is break. With the right suction applied to a cell, you can actually suck a hole into the membrane. Often, it only takes a gently, quick “kiss” to do so. At that moment, the liquid inside your capillary and the liquid inside the cell will mix. That’s why it is so important to know what to put inside the capillary – now, latest, it will determine life, death and function of your cell.
What you basically have now is a connection to the inside of the cell. This is the one true way to measure a potential “across” the membrane. A potential is a difference in electricity between two places. With your one wire connected to the inside of the cell and another one outside in the liquid, you can measure the potential between the inside and the outside of the cell.
Now we’ve got the patch, but where is the clamp?
So, being in such a position, what will you actually see when you connect an electricity-reading instrument to your wire in the fluid-filled capillary?
Nothing.
How much electricity do you think a cell generates? For example, the “electricity” (a potential, actually) that you use from your outlets is 110 Volts (220 Volts for some countries). The maximal current you should demand from your outlet at once is 16 Amperes, then the fuse will blow. If your cells generated that much energy, they’d not survive. Neither would your electrophysiology equipment, but let’s not go there. The range of a nerve cell is more in the “mV” area. In fact, many nerve cells have a constant potential that is somewhere between -50mV to -90mV. By letting ions flow through their membranes by channels or gates or even pumps, they generate currents from several “pico-Amperes” (1 pA = 0.000000000001 Amperes) to several nano-Amperes (that’s three zeros less). This can’t just be measured – you need to amplify the signal to actually be able see it.
Therefore, every “patch clamp setup” (that is a microscope with all the equipment needed for the technique) will always include two amplifiers. One is connected right to the patch pipette, on top of the manipulators. It serves as a holder for the pipette and is connected to the wire inside. This “pre-amplifier” is connected to a huge box, the amplifier, near your computer that does the final amplification. If you now connect an oscilloscope to this amplifier, you can literally watch the cell’s electrical activity. The cursor, just like a heart-monitor in a hospital, will jump up and down across the screen and draw a line symbolizing the electrical activity of the cell.
If you want to save these data to a computer, you’ll also need is a digitizer inbetween your amplifier and your computer. That is because the amplifier generates an analogue signal that doesn’t go with our base-10 digital system that your computer relies upon. The digitizer converts the analogue, amplified signal from the cell to a form the computer can read.
Yet, the cell is not clamped!
Well, if the computer can read a signal, why not give a signal as well? That’s actually possible through the digitizer, which also works the other way round. Usually, a cell that is left alone or connected to others will spontaneously do at least something. Occasionally there’ll be crass changes in electrical potential or changes of current flow or even constant repetitive activity, especially when your neuron is connected to others.
However, you can now also use the wire in the pipette to temper with the cell and change it’s potential or “inject” current into the cell and it has to deal with that. Modern patch clamp set up have smartly constructed mechanisms that are able to keep the cell at a certain potential and compensate for any activity it might spontaneously have – more or less quickly. When you decide to “keep” your cell at a certain potential, you’re “clamping” it to that potential. For example, there are channels in nerve-cell membranes that are active or inactive at certain potentials. If you want to find out about a particular channel or stop it from disrupting your measurements, clamping a cell to a certain potential might be a smart thing to do.
It’s also often useful, if you want to compare data, to keep all your cells on a specific potential, like -70mV. You can further “feign” activity or force the cell to change to a certain potential and see how it reacts to that. Instead of just listening in on the radio station, you’re now tempering with the music they play, though your possibilities are limited by the record collection in their shelves. For example, cells that unlike nerve cells don’t have the ability to generate what is called an “Action Potential” cannot be forced to do so because they simply lack the equipment.
Nerve cells, however, do generate action potential and when you gradually, step-by-step change the “potential” of the cell from -70mV to -65mV to -60mV and so on (making the potential more positive is called “depolarizing”), at some point the cell will generate such action potentials:
Voltage Clamp and Current Clamp
Clamping your cell to a certain potential is called “Voltage Clamp”. What you measure in Voltage Clamp is the current that you need to inject or subtract to keep the cell on your desired voltage. For example, if your cell releases positively charged ions from inside the cell to the outside, the inside of the cell becomes more negative. You’ll need to inject a positive current to keep it on your desired level and that current is what you will measure.
The Current Clamp is not an actual “clamp” like the voltage clamp. Instead of deciding which potential to keep your cell at, you simply decide how much current you wish to constantly inject into the cell (usually a few picoAmpere). Usually, this leads to the same result, though – if you constantly inject the same current, you’ll keep a healthy cell on a certain potential. Only if the cell spontaneously changes its potential, you don’t compensate, you just keep injecting the same current and let the cel do. What you measure when injecting current is the change in potential (in mV). In the Voltage Clamp, you decide the voltage and measure the flow of current across the membrane (pA or nA). The most used variant of the Current Clamp, often called CCfast, is one where you actually DO clamp the potential (either by adjusting the current by hand to keep your cell on the desired potential or by letting the computer do it). It’s truly a bit confusing because in a way, CCfast is a Voltage Clamp mode.
Altogether, the difference between CC and VC are not so important to a lay person. In short it can be said that in CC, the cell has more freedom to do as it pleases and it is the “watch mode” for the electrophysiologist. The Voltage Clamp has the advantage that spontaneous activity of the cell is quickly compensated for so that it can’t go crazy and do as it pleases – it’s the “control mode”. It’s useful to watch small events that you don’t want to be overshadowed by larger activity.
Inside Out, Outside In?
You can actually go even one step further than opening up the inside of the cell. By pulling the capillary backwards and employing the right use of air pressure or suction, one can actually isolate a membrane patch. This is either done so that the membrane closes as it was before or – because membranes like to stick to each other – so that it turns inside out (I reposted the above picture to show that). The first is the “inside out” patch, because what used to be the “inside” side of the cell is now outside of the capillary. The second variant is the “outside out” patch, where the former outside of the patch is now outside of the patch pipette.
No matter which version you use, there’s a certain chance that your pipette will be stuck over one single ion-conducting channel in the membrane. In that case, you might be able to witness the repeated opening and closing of one single channel. I find that that is pretty cool.
The hardship of the young patch clamper
By the way – this whole explanation here is still way easier than it sounds. To just name a few problems that already go to far: What you record isn’t always coming from the cell. One reason you have a second electrode outside of the cell in the bath is to know what electrical “noise” is already there and to subtract it from your signal. It would be nice if this was the only “noise”, but such things as fridges plugged into an outlet three rooms down the corridor, or lamps picking up electrical waves from some technical equipment in the room next to you are only some of the noise-sources. Since you know an outlet has over 1000 times more potential than your cell, you can imagine how your signal simply drowns if only a fraction of that “noise” reaches your sensitive measuring device. In that case, you need to search for the source and eliminate the problem.
As sensitive as the amplifiers are – especially the small pre-amplifier – even the little electrical shock you may get from your car or your cat on a hot summer day could completely destroy your pre-amplifier.
In fact, to avoid such expensive damage, the entire microscope is usually mounted on a metal plate that is connected to the amplifier’s “ground” outlet that much like a lightning conductor moves all electricity out of harm’s way. By touching the metal plate, you can avoid damaging your pre-amplifier because you’re now also “grounded”. However, if only lamps and tables and shared outlets picked up such noises it would be nice. Any electrophysiologist will tell you about noises and noise-sources and probably swear that it’s all some mysterious voodoo. Broken cables, air conditioning systems and fans, metal screws and crossing, isolated cables are just a few creative examples for noise sources – but they don’t necessarily have to be. The detection of noise and its removal are quite an art as every electrophysiologist will happily confirm to you. In many cases, some protection can be achieved by wrapping your setup in a farraday cage and cables in aluminum foil, but that’s not always certain.
Other sources of annoyance are patch pipettes that for some reason (a little strain on a cable, a loose screw…) aren’t standing still and “drifting” away from your cells. Or any sort of physical impact that moves your microscope like a miniature earthquake and separates cells form patch pipette. To avoid that, the metal plate under the microscope is usually mounted on an air-pressure table that cushion such impacts. Much testing and correcting is also done with the composition of liquids, osmolarity and pH-values to make the cells willing to patch or seal closely with the capillary and ensure their viability. There are currents leaking out of your capillary, and potentials generated between your wire and your solution or between your membrane and the inside of the cell that need to be calculated out of the signal of compensated for through the amplifier.
In short, things are not nearly as simple as they seem, but when are they ever? Getting a nice, clear signal without noises and interruptions and stable, healthy cells or tissues are the dream of any electrophysiologist. In many cases, an acceptable option has to be preferred over the perfect one, but as equipment and people are getting smarter and smarter about the bits and tricks of electrophysiology, measurements get finer and finer and more sophisticated.
However
Once your cell is sealed and the membrane patch is broken, you become the witness of one of the most amazing phenomenons our own bodies have to offer – natural, electrical communication – action and reaction – the whisper in the neuronal network! The little world of a single cells! And not only can you listen to it, you can add your own share via the knobs you twiddle on your amplifier, drugs you apply in the bath or the patch pipette and maybe even DNA you offer to your cell. It’s like sticking a bug into a strange, different world, and becoming part of it for just a small while.
Electrical currents, reactions, actions and properties can tell an electrophysiologist a lot about a cell – the rough number of connections, maybe, their health, the nearby composition of channels and their willingness to get excited. But also changes in electrical properties are very helpful tools and interesting subjects. Many electrophysiologists themselves will get very excited staring at those little curved and straight lines on their computer or oscilloscope. I admit, it must seem odd to an outsider!
There is much that can be done with this, but for now it would go to far. 
Just keep in mind what an amazing technique we’re talking about here: A device to listen in on the very small and very quiet world of the electrical activity of a single cell or a single channel on a cell’s membrane!
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