History of electrophysiology and the patch clamp article pdf available in methods in molecular biology clifton, n. Today, the patchclamp technique is the main technique in electrophysiology to record action potentials or membrane current from isolated cells, using a patch pipette to gain electrical access to the cell. Patch clamp is the gold standard technique for highfidelity analysis of the electrical properties and functional connectivity of neurons. Robotic multiwell planar patchclamp for native and primary. Links to low cost no cost electrophysiology software. Patch clamp recordings in the whole cell configuration, and single channel recordings in the cell attached configuration, can be performed with planar patch clamp chips. The main feature of gap junctions is the passage of ions and small molecules between adjacent cells. To measure the membrane potential, the multiclamp 700b and axoclamp 900a both monitor voltage drop initiated by current injection along an inseries resistor. They move your patch pipette with nanometer precision. Ppt the patch clamp technique powerpoint presentation. Patch clamping aims to cover the basic principles and practical applications of this important technique. Abstract wholecell patchclamp techniques are widely used to measure membrane currents from isolated cells. Sakmann and neher develop the patch clamp technique in 1970s and early 1980s.
Again, this is great if one is already practicing patch clamp and is looking to expand ones skill set. Nanion portapatch planar patch clamping automate scientific. Here we show the wider potential of the multiwell approach with the capability for efficient intracellular solution exchange, describing protocols and success rates. We present a technique which combines two methods in order to measure the series resistance r s during wholecell patchclamp recordings from excitable. It helps in currentclamp recordings, field potential recordings in ion channel and highspeed current clamp recording within the same head stage. The patch clamp technique was originally developed in the late 1970s 25 and further improved by hamill et al. Pdf history of electrophysiology and the patch clamp. Understand whole cell capacitance and series resistance hello. In voltage clamp, series resistance prevents your amplifier from charging the membrane capacitor, and in current clamp, series resistance stops your cell from being able to charge the capacitance of your pipette. Oetiker ear clamps license plate fasteners flange bolts 10. By carefully heating and pulling a small glass or quartz capillary tube, a very fine pipet can be formed.
Patch clamp recording instruments with a choice of 3 headstages are suitable for measuring currents in whole cell measurements and single channel studies. Fasteners vacuum connectors metric hoses metric clamps wheel weights. An introductory guide to patch clamp electrophysiology a last chance effort. One i break into my cell, i clamp and 60mv and input a pulse of 4mv. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. Planar patch clamp is a novel method developed for high throughput electrophysiology. Library for reading files created by heka pulse and patchmaster. The patch clamp technique allows the investigation of a small set or even single ion channels. The dagan 3900a patchwhole cell clamp breaks new ground with a new concept.
The intan clamp system allows users to perform single amplifier or multiamplifier patch clamp electrophysiology experiments with small, affordable hardware and free, opensource software. An introductory guide to patch clamp electrophysiology is a concise introduction to the basic principles and practical applications of this important technique. Stainless steel clamps can be installed above or below ground and are available for all types of pipe, with. These studies established the physiological importance of ion fluxes through ion channels in the activity of cells and set the foundation for much of the subsequent work in.
Wholecell patchclamp techniques are widely used to measure membrane currents from isolated cells. Can read a trace from a whole group, series or or a sweep. I recorded mitral cells using whole cell patch clamp. The traditional manual method to patch clamp using glass pipettes was developed by erwin neher and bert sakmann and required a highly skilled technician. This device connects to a host computer through a usb 2. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the wholecell configuration. World precision instruments amplifiers patch clamp ipa. Axopatch patch clamp amplifier is the premier microelectrode amplifier for ultralow noise patch clamp recordings. Patch clamp technique an overview sciencedirect topics. Worlds best powerpoint templates crystalgraphics offers more powerpoint templates than anyone else in the world, with over 4 million to choose from.
I then put on wholce cell capacitance to reduce the fast component of my transients the slow components remain. Development of planar patch clamp technology and its. A prerelease patch clamp electrophysiology software written in matlab uses national instruments m and x series boards. Patch clamp techniquethe patch clamp technique is a laboratory techniquein electrophysiology that allows the study of single ormultiple ion channels in cells. Extracellular, intracellular and patch clamp amplifier. The interpretation of currentclamp recordings in the cell. Furthermore, the extracellular membrane remains accessible for compound application during the experiment. It can reach up to 10100 g meanwhile, the distance between the microelectrode and the cell membrane is less than 1 nm, so that the membrane patch is electrically isolated from the surroundings fig. Clamp patch clamp amplifier system intan technologies. Measurement of cellular excitability by whole cell patch. To increase bandwidth, it is necessary to compensate for r s. Included are i the wholecell patch technique using either small or largepore suction pipette electrodes to record wholecell i na or i gna, ii an oilgap voltage clamp that utilizes a 30 to 40mwide oil gap to electrically isolate the voltageclamped end of a single cell from the other end that has had its cell membrane physically. The classical biophysics of the nerve impulse had by then been established in the squid giant axon using a combination of 1 voltage clamping with axial wire electrodes and 2 internal. In patch clamp experiments, suction is used to attach a micropipette filled with electrolyte solution to the cell membrane.
This forms a seal, isolating a patch of the membrane to enable the flow of currents across this section of the membrane to be measured. The common recording modes of the patchclamp technique are current clamp and voltage clamp. Combined with optical measurements, the patch clamp chip will facilitate biophysical and neurobiological research on ionic channels, and will promote the drug discovery. They are used with various headstages, suitable q series electrode holders and glass microelectrodes purchased separately to measure corresponding currents in single channel measurements or whole cell studies. Feb 22, 2012 patch clamp techniquethe patch clamp technique is a laboratory techniquein electrophysiology that allows the study of single ormultiple ion channels in cells. The patch clamp amplifier thus must function as a currenttovoltage converter to allow this. The whole cell patch clamp can operate in two recording modes depending on the configuration of a patch clamp amplifier. Traditional patch clamp has a very low throughput which has been proven to be a. To measure whats happening in or on a single, living cell, scientists use a technique called the patch clamp which requires an extremely fine pipet held tightly against the cell membrane. Using a pipette puller such as a sutter p97 that produces two identical sister tips from the same piece of glass is advantageous when multiple drug solutions are to be used on the same cell. This is quite simply the best intro to patch clamp i have read. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic. Applied in cell culture, this technique provides accurate control of the. Fully integrated patch clamp amplifier and data acquisition system ensures quick and easy setup optimized for wholecell patch clamp recordings in tissue slices, adherent or dissociated cells.
These things are bad, but what is worse is when it changes over time. Other resistivefeedback headstages are also separately available for use with the patch clamp amplifier. With the latest developments in the traditional patch techniques such as wholecell and single channel as well as perforated patch, fast drug application, loose patch and macropatch. In the voltage clamp mode the recorded signal is the transmembrane current and the controlled input is the clamped membrane voltage delivered to the cell examined. I am new to patch clamping and wantd to ask about whole cell capacitance and series resistance. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible.
Winner of the standing ovation award for best powerpoint templates from presentations magazine. In general, patch pipettes are used to electrically isolate and study. Oct 24, 2012 introduction the patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells. The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. In addition, the patch clamp technique has become a powerful method for investigating the mechanisms underlying the effects of acupuncture. Several patch clamp configurations can be used depending on the research interests, but in all cases, electrophysiological recordings are produced using a glass micropipette in contact with a patch of the neurons membrane.
Internal perfusion can be used not only for patch clamp. Whole cell patch clamp recordings from morphologically digitimer ds2a duration. Tightseal wholecell and cellattached patchclamp recordings in voltage and currentclamp mode were carried out using an axopatch 200a amplifier axon instruments, union city, ca. Patch pipettes the patch clamp technique is used in electrophysiological research to study the electrical activity of neurons at the cellular level. Cellular and molecular events can be investigated using electrophysiological techniques.
Microfluidic system for planar patchclamp electrode arrays. This section is designed for users that cant wait to get something done with patchmaster. These workhorse patch clamp amplifiers by warner instruments are ideal equally for research and teaching applications. Microfluidic system for planar patchclamp electrode arrays xiaohui li yale university 2006 the patch clamp has been widely accepted as a standard technique for fundamental studies of ion channel proteins, and discovery of drugs that affect these proteins.
Planar patch clamp article about planar patch clamp by the. The procedure involves pressing a glass micropipette against a cell in order to isolate a small patch of membrane that contains one or more ion channels. A patch clamp chip can form a high seal resistance automatically, and performs high throughput cellular electrophysiology recordings. They are used to repair holes, leaks, splits, circumferential breaks and cracks, as well as to correct pulled or leaking service connections. Patch clamp electrophysiology is used to study the electrical properties of excitable cells and ion channels. Of course, you will not have to do a real experiment, instead you should use the model circuit to simulate the conditions of a patch clamp. Axon instruments patchclamp amplifiers molecular devices. A single ion channel conducts around 10 million ions per second. Continuing the research of the bestselling first edition, patch clamp analysis. The patchclamp technique was originally developed in the late 1970s 25 and further improved by hamill et al. Theyll give your presentations a professional, memorable appearance the kind of sophisticated look that todays audiences expect. In particular, the patch clamp method provides detailed information.
In order to carry out my protocol, i need the whole cell configuration to remain stable for relatively long periods of time in voltage clamp. Whereas conventional headstages measure pipette current as the voltage across a resistor, the headstage of the 3900a integrates the current on a capacitor, then differentiates the charge signal into a current signal. While suitable for a broad range of ionic currents, the series resistance r s of the recording pipette limits the bandwidth of the wholecell configuration, making it difficult to measure rapid ionic currents. Patch clamp manipulators are highly precise instruments. Fittings auto bulbs and harnesses electrical shop supplies cotter pins. Library for reading files created by heka pulse and patch master. Understand whole cell capacitance and series resistance.
Pull a standard patch clamp micropipette as described above. Historically the patchclamp technique was based on the work of alan hodgkin and andrew huxley who, in the 1950s, conducted a series of elegant voltageclamp experiments that allowed the recording of macroscopic currents in the squid giant axon by controlling the voltage of the membrane hodgkin and huxley, 1952. Several patch clamp configurations can be used depending on the research interests, but in all cases, electrophysiological. In this paper, recent researches on how acupuncture might modulate electrophysiological responses. In wholecell voltageclamp mode 7075% series resistance compensation was achieved using the series resistance compensation feature of the 200a amplifier. Testing silicon planar patchclamp devices simeon bamford, keith baldwin and evangelos delivopoulos, supervised by john curtis and nikki macleod neuroinformatics doctoral training centre, university of edinburgh school of electrical engineering, university of edinburgh department of biomedical sciences, university of. Can someone advise on series resistance in current clamp. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in.
The technique requires using a blunt pipette with a 34mm short taper and a. Advanced techniques, second edition collects three more years of research in the everexpanding study of the cell membrane. Wholecell patchclamp recordings for electrophysiological. Jun 08, 2012 whole cell patch clamp recordings from morphologically digitimer ds2a duration. Abstract wholecell patch clamp techniques are widely used to measure membrane currents from isolated cells.
The wholecell mode of the planar patchclamp technique portapatcho nanion, munich. The technician would position the glass pipette near a cell and apply the appropriate suction to create an electrical seal between the pipette and the cell membrane. The model 2400 is a lownoise, fullfeatured patch clamp amplifier designed for voltage clamping using patch electrodes on single channels or whole cells. Faraday cage heka table top faraday cage tmc benchtop faraday cage the cage around your patch clamp rig shields noise from interference sources in order to get a perfect signaltonoise ratio. Received the nobel prize for this high scientificwork in1991.
Series resistance compensation for wholecell patchclamp. Portapatch planar patch clamping the portapatch is a onecellatatime system, which is surprisingly small for a complete patch clamp rig. Dynamic clamp as a tool to study the functional effects of. It brie y describes how a very simple rst experiment with patchmaster could look like. Instead of positioning a pipette on an adherent cell, cell suspension is pipetted on a chip containing a microstructured aperture. Patch clamp electrophysiology, voltage clamp, action potential. Offers realtime measurements of neuronal cell function and communication. Data collection is initiated through a rapid parameter directory rpd which maintains a list of 18 parameter filenames or script filenames. While suitable for a broad range of ionic currents, the series resistance r s of the recording pipette limits the bandwidth of the wholecell configuration, making it difficult to. Jul 05, 20 i am new to patch clamping and wantd to ask about whole cell capacitance and series resistance. The principal concerns of modern electrophysiology are 1 the physicochemical processes on the cell membrane that give rise to electric potentials and changes therein during active physiological processes, 2 the biochemical processes that supply energy for the transport of ions across membranes and for the creation of ion gradientsthe. In the heart, for example, junctional currents between adjacent cells are elicited by the membrane potential difference when one of the cells generates an action potential, while the other cell is still at resting membrane potential, thereby allowing propagation of the action potential. Wavesurfer developed at hhmi by the svoboda, magee, spruston jayaraman and koyama labs.
Amplifier for voltage clamping using patch electrodes on. At all times the patch clamper can determine cell status. Its design also allows fast intracellular current clamp measurements with sharp electrodes. Fully integrated patch clamp amplifier and data acquisition system ensures quick and easy setup optimized for wholecell patch clamp recordings in.
The technique can be applied to a wide variety ofcells, but is especially useful in the study of excitablecells such as neurons, cardiomyocytes, muscle fibersand pancreatic beta. Microelectrodes or glass electrodesmicropipettes are not supplied. With membrane potential fixed, the membrane current is measured. The technique of patch clamping can be seen in retrospect as a combination of two separate lines of development that both originated in the 1960s and 1970s. The key point of patch clamp is to form gigaohm seal resistance between a microelectrode and a cell membrane. An introductory guide to patch clamp electrophysiology a last chance effort before giving up on finding a good patch clamp book. Determination and compensation of series resistances during whole. Patchclamp is the gold standard technique for highfidelity analysis of the electrical properties and functional connectivity of neurons. A single cell is then positioned on the hole by suction and a tight connection gigaseal is formed. Local application of drugs to study nicotinic acetylcholine. During a patch clamp recording, a hollow glass tube known as a micropipette or patch pipette filled with an electrolyte solution and a recording electrode connected to an amplifier is brought into contact with the membrane of an isolated cell. The reader should not worry about options that are unclear, because more detailed descriptions of all of the mentioned steps are to follow. Others such as patchclamp methods and protocols are just that. A variety of intraand extracellular recording techniques, both tissue slices and in vivo electrophysiology, coupled with discrete electrical stimulation and drug iontophoresis, can provide important mechanistic insight into drug action on ion channels, synaptic transmission and plasticity, and intrinsic excitability.
1410 84 946 137 1147 1558 788 1449 816 315 1431 1174 39 1234 639 308 1515 797 1083 1239 73 1385 980 323 1087 1245 674 393 758 262 1365 723 954 153 1309 503 582