Electrophysiology
Home of the famous Axon Guide:
A guide to Electrophysiology and Biophysics Laboratory Techniques
What is electrophysiology?
Electrophysiology is the field of research studying current or voltage changes across a cell membrane.
Electrophysiology techniques are widely used across a diverse range of neuroscience and physiological applications; from understanding the behavior of single ion channels in a cell membrane, to whole-cell changes in the membrane potential of a cell, to larger scale changes in field potential within the brain slices in vitro or brain regions in vivo.
Patch-clamping, one of the most widely used electrophysiology techniques, is the best tool to study the activity of ion channels, which are major targets for researchers due to the key role they play in many neurological and cardiovascular diseases, as well as their physiological functions.
Extracellular field-potential recording technique can be used to study the synaptic activity of a population of neurons, and it can help us understand how information is processed in the brain.
Electrophysiology lab
Each electrophysiological labratory setup is different, reflecting the requirements of the experiment or the foibles of the experimenter. Here we describe components and considerations that are common to all setups dedicated to measure electrical activity in cells. An electrophysiological setup has four main lab requirements:
- Environment — the means of keeping the preparation healthy;
- Optics — a means of visualizing the preparation;
- Mechanics — a means of stably positioning the microelectrode; and
- Electronics — a means of amplifying and recording the signal.
The illustration below shows a standard electrophysiology rig setup – a table and cage to shield your setup from external interference; microscope with micromanipulator for stably positioning the microelectrode; an amplifier to collect and amplify the acquired signals; a digitizer to convert the analogue signals into digital signals, and data acquisition and analysis software, to both set up the experimental protocols and extract meaningful, actionable results from the data collected.
Electrophysiology solution: Axon equipment
The Axon™ instrument portfolio provides a comprehensive solution for patch-clamping that includes amplifiers, digitizer, software, and accessories. Our best-in-class instruments facilitate the entire range of patch-clamp electrophysiology techniques from the smallest single-channel to the largest macroscopic recordings.
The Axon pCLAMP™ 11 Software Suite is the most widely used electrophysiology data acquisition and analysis program for control and recording of voltage-clamp, current-clamp, and patch-clamp experiments. Several key features listed below help streamline workflow, allowing you to do more sophisticated experiments, execute them more efficiently and generate higher quality data.
AMPLIFIER(S)
What is an patch clamp amplifier? An instrument that contains the circuitry required to measure electrical currents passing through ion channels or changes in cell membrane potential.
Why use it? To measure changes in current or voltage. The amplifier contains the circuitry necessary to measure current passing through the cell membrane both in magnitude and direction.
The amplifier can also measure the cell membrane potential in response to the movement of current. To initiate current movement, the experimenter can deliver a voltage command to the cell, and the cell will respond by passing the current necessary to maintain that voltage command. Conversely, the experimenter may also inject current and then measure the change in membrane potential resulting from that change in current. Choosing where to amplify and filter the signal of interest has implications on signal fidelity. The ideal place to amplify the signal is inside the recording instrument. All models of Axon™ amplifiers use this strategy with variable gain control on the output to provide low-noise amplification of the pipette current or membrane potential. Placing the amplification inside the recording instrument minimizes the amount of circuitry between the low-level signal and amplifying circuitry reducing extraneous noise sources.
Available amplifiers: Axopatch™ 200B, MultiClamp™ 700B, Axoclamp™ 900A
DIGITIZER(S)
What is it? The digitizer is a data acquisition instrument that converts analog signals into digital signals.
Why use it? Digitizers capture data for analysis.
The current acquired by the amplifier is an analog signal, but in order to perform data analysis needed for high resolution patch-clamp measurements, the analog signal must be converted into a digital one. Positioned between the amplifier and the computer, the digitizer accomplishes this important task. The signal quality the computer receives is extraordinarily important, and this is determined by the sampling frequency, or sampling rate. The latest generation of Digidata® digitizers has the capability of sampling at 500 kHz and is equipped with the HumSilencer™ feature, which can eliminate 50/60 Hz line-frequency noise.
Available amplifiers: Digidata 1550B Low Noise Data Acquisition System plus HumSilencer
SOFTWARE
What is it? Patch clamp data and acquisition and analysis software is your interface with the amplifier, digitizer, and any other patch-clamp electronics.
Why use it? To perform data acquisition and data analysis, as well as to control the digitizer and amplifier.
While the amplifier and digitizer together hold the key circuitry that implements a patch-clamp experiment, the software controls these instruments so they deliver the desired potential(s) and measure the resulting current or voltage. In addition, the software analyses the acquired signal with user-defined settings, which can include filtering, normalization, noise removal, curve fitting, and parameter determination.
Available amplifiers: pCLAMP™ 11 Software
HEADSTAGE
What is it? A device that holds the micropipettes with built-in circuitry to transmit electrical signals from the micropipettes onto the amplifier.
Why use it? The electrical signal acquired by the micropipette needs to be transmitted to amplifier systems for signal processing.
Each headstage is specifically tuned for the amplifier. All headstages contain critical electric circuitry that reduce noise. The headstage is also mechanically controlled by the micromanipulator.
Available headstages: Axon headstages
MICROSCOPE WITH MICROMANIPULATORS
What is it? The microscope is an optical magnification tool. The micromanipulator is a device that mechanically maneuvers the micropipette with nanometer precision, typically allowing 3-dimensional movements.
Why use it? To precisely and stably position the micropipette to the area of cell membrane, which is critical for successful recording.
Accurately placing a patch electrode onto a 10-20 µm cell requires an optical system that can magnify up to 300- or 400- fold with contrast enhancement (e.g. Nomarski/DIC, Phase, or Hoffman) and a micromanipulator that stably positions the electrode in 3D space. An inverted microscope is preferable because it allows easier access for electrodes from above the preparation and also provides a larger, more solid platform to bolt the micromanipulator. A micromanipulator has the ability to move the electrode in very minute distances along the X, Y, and Z axes. The micromanipulator can then hold that position indefinitely.
FARADAY CAGE AND AIR/ANTI-V
What is it? A table and cage around your patch-clamp setup to isolate sources of interference.
Why use it? To shield your setup from external interference.
Electrical currents measured during patch-clamp experiments can be extremely small (in the pico-amp range), and any small sources of interference, such as radio waves, can distort or obscure these signals. A Faraday cage is a wire mesh enclosure around your microscope and recording chamber; it is useful in preventing the electrodes from picking up extraneous noise sources. Additionally, small sources of vibration on the order of pico-meter magnitude can disrupt your recording. Hence, all components must be perfectly positioned throughout the time-course of your experiment, and the air or anti-vibration tables are used to isolate your setup from external sources of vibration that may disrupt this alignment.
