Patch clamp electrophysiology
Electrophysiology is one of the foundational disciplines in neuroscience and cardiac physiology for the evaluation of ion channels. The Patch-clamp technique is a versatile electrophysiological tool for understanding ion channel behavior.
Every cell expresses ion channels, but the most common cells to study with patch-clamp techniques include neurons, muscle fibers, cardiomyocytes, and oocytes overexpressing single ion channels. To evaluate single ion channel conductance, a microelectrode forms a high resistance seal with the cellular membrane, and a patch of cell membrane containing the ion channel of interest is removed. Alternatively, while the microelectrode is sealed to the cell membrane, this small patch can be ruptured giving the electrode electrical access to the whole cell. Voltage is then applied, forming a voltage clamp, and membrane current is measured. Current clamp can also be used to measure changes in membrane voltage called membrane potential. Voltage or current change within cell membranes can be altered by applying compounds to block or open channels. These techniques enable researchers to understand how ion channels behave both in normal and disease states and how different drugs, ions, or other analytes can modify these conditions.
Techniques of Electrophysiology
What is continuous single-electrode voltage clamp (cSEVC)? It is an electrophysiology patch-clamp method that passes a membrane voltage into a cell and measures the change in current as the voltage steps.
An action potential is a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane with a characteristic pattern. Examples of cells that signal via action potentials are neurons and muscle cells.
The current or voltage signal acquired by the amplifier is an analog signal, but 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. Signal quality is extremely important and is impacted by the sampling frequency. The latest generation of Digidata digitizers sample at 500 kHz and can be equipped with HumSilencer, which eliminates 50/60 Hz line-frequency noise.
Single Channel Recording
The patch-clamp technique involves a glass micropipette forming a tight gigaohm seal with the cell membrane. The micropipette contains a wire bathed in an electrolytic solution to conduct ions. To measure single ion channels, a “patch” of membrane is pulled away from the cell after forming a gigaohm seal. If a single ion channel is within the patch, currents can be measured. The Axopatch 200B, with extremely low-noise profile, is ideal for this application, maximizing signal for the smallest conductance ion channels.
Action Potential Search
Action potentials represent important cellular events. Without action potentials, hearts would not beat, and neurons would not fire, so measuring these events is essential. The Action Potential Search tool in Clampfit 11 Advanced module detects all action potentials in the data file. It applies user defined and programmatically determined metrics including amplitude, AP duration, rise and decay time, rise and decay slope, peak to peak frequency and time, amplitude delta per peak, afterpotential amplitude and duration, and threshold potential.
An ion channel is a group of proteins that form a pore across the lipid bilayer of a cell. Each channel is permeable to a specific ion (examples: potassium, sodium, calcium, chloride). Patch-clamp is used to evaluate current or voltage in the membrane associated with ion channel activity via direct measurement in real time using ultra-sensitive amplifiers, high-quality data acquisition systems, and powerful software to evaluate the results.
The patch-clamp technique involves a glass micropipette forming a tight gigaohm (GΩ) seal with the cell membrane. The micropipette contains a wire bathed in an electrolytic solution to conduct ions. The whole-cell technique involves rupturing a patch of membrane with mild suction to provide low-resistance electrical access, allowing control of transmembrane voltage. Alternatively, investigators can pull a patch of membrane away from the cell and evaluate currents through single channels via the inside-out or outside-out patch-clamp technique.
Whole Cell Recording
The whole cell patch-clamp technique involves a glass micropipette forming a tight gigaohm (GΩ) seal with the cell membrane. This micropipette contains a wire bathed in an electrolytic solution to conduct ions. A patch of membrane is subsequently ruptured by mild suction so that the glass micropipette provides a low-resistance access to the whole cell, thereby allowing the investigator to control the transmembrane voltage and allowing the investigator to evaluate the sum of all currents through membrane bound ion channels.
Series Resistance Compensation
Series resistance is the sum of all resistances between the amplifier and the inside of the cell using the whole-cell recording method. Due to Ohms Law, the larger this resistance, the greater the difference between the command level and the measured values. This creates an error in actual voltage or current measurement potentially leading to inaccurate observations. To overcome this, the Molecular Devices amplifiers have built-in circuitry to improve the bandwidth of the recording by compensating the error introduced by the voltage or current drop across the series resistance.
Voltage Clamp Amplifier
In an experiment using the voltage-clamp method, the investigator controls the membrane voltage in a cell and measures the transmembrane current required to maintain that voltage. This voltage control is called a command voltage. To maintain this command voltage level, an amplifier must inject current. The current injected will be equal and opposite the current escaping through open ion channels, allowing the amplifier to measure the amount of current passing through open membrane bound ion channels.
Current Clamp Amplifier
Current-clamp is a method used to measure the resulting membrane potential (voltage) from an injection of current. To measure the membrane potential, the MultiClamp 700B and Axoclamp 900A both monitor voltage drop initiated by current injection along an in-series resistor. Current-clamp is commonly used to inject simulated, but realistic current waveforms into a cell, and monitor membrane effect. This technique is ideal for the evaluation of important cellular events such as action potentials.
50/60 Hz line-frequency noise, also known as electrical hum, is the most common source of background noise in patch-clamp electrophysiology experiments. This noise can overwhelm biological signals of interest, making sensitive patch-clamp measurements nearly impossible. Traditional trouble shooting is typically only partially effective and can impair data accuracy. HumSilencer is a filter-free, adaptive technology that learns and removes line frequency noise without using methods that impair signal accuracy like filters, that can distort biological signals.
Cellular Pathway Analysis
Ion channels are involved in many cell pathways and understanding the function of ion channels in response to changes in membrane potential or the presence or absence of other molecules is important in order to understand exactly how ion channels participate in normal and abnormal biological processes such as cell differentiation and migration, disease states, and neuronal communications.
Ion channels play a role in many diseases including hypertension, cardiac arrhythmias, gastrointestinal, immune and neuromuscular disorders, pathological pain, and cancer. By understanding the exact role that ion channels play in a particular disease, researchers might be able to find a way to affect the ion channel in such a way as to alter the course of the disease.
Population Spike Search
Population spike recordings and paired-pulse experiments, while simple to collect, have traditionally been difficult to analyze. That is no longer the case with the Clampfit Advanced Analysis Module in pCLAMP 11 Software. The Population Spike Search tool will automatically locate population spikes based on user defined parameters and calculate the amplitude, area under the curve, half-width, rise time, decay time, rise slope, decay slope, and coastline of population spikes and paired-pulses.
Batch Data Analysis Macros
Clampfit Advanced Analysis Module, part of the pCLAMP 11 Software suite contains a Batch Data Analysis Tool that utilizes macros to accelerate data analysis. Batch analysis saves time by analyzing abundant amounts of data created by the same protocol. To use batch analysis, simply turn on the macro capture feature, analyze the data, and save the macro. When additional data needs analyzing, simply apply the saved macro and the data is analyzed automatically.
Automated Event Detection
Clampfit Advanced Analysis Module, part of the pCLAMP 11 Software suite, has a flexible event detection engine that analyzes spontaneous and evoked action potentials and post-synaptic data. Events are detected by threshold crossing or a pattern-matching Template Search. Template Searches analyze spontaneous events like miniature synaptic EPSPs and IPSPs. Additionally, multiple event categories of events can be simultaneously detected. The integrated environment of Clampfit 11 Software links detected events in data to the spreadsheet and graph windows enabling quick contextual evaluation of the entire dataset.
Resources of Electrophysiology
Videos and Demos
How to Combine Traces, Calculate Rise or Decay Time Constant, and Perform Curve Fitting Using Axon pCLAMP Software
How to Create Customized Command Waveforms Using the pCLAMP Software
The Use of Sequencing Keys, User List, and Stimulus File with pCLAMP Software
Synchronizing Electrophysiology and Imaging Solution with Axon pCLAMP and MetaMorph Software
Online Statistics, Membrane Test Between Sweeps in Clampex and Analysis of Synaptic Events with the Clampfit™ Data Analysis
Use of the Axoporator 800A for Single-cell Electroporation for Transfection and Dye-labeling
Using the Axoclamp 900A for Two-Electrode Voltage-Clamp of Xenopus Oocytes Expressing Ion Channels
Writing Long-Term Potentiation and Depression Protocols and the Use of Filters in Data Acquisition and the Clampfit Application
Series Resistance Compensated or Not
Using Electrophysiological Studies to Accelerate Mechanistic Study in Reception and Transmission
Update and Hardware Choices for Optogenetics Considerations for Synchronized Light Patterning
Investigations of the Effects of Amyloid-Beta Proteins on hSlo1.1, a BK Channel, in a Xenopus Oocyte Model
Nanopores-Electronic Tools for Single-Molecule Biophysics and Bio-Nanotechnologies
Axon Amplifiers and pCLAMP Software-key Features Reviews (Chinese version)
Basic Single Channel Analysis Using Clampfit
Action Potential Analysis in Clampfit Module
A Walkthrough of the Protocol Editor in the pCLAMP Data Acquisition Module
A Walkthrough of Protocol Editor in pCLAMP (Chinese version)
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