The Planar Lipid Bilayer is an ideal tool for measuring pico- or nano-scale charge currents across a membrane.
This page generally describes a Planar Lipid Bilayer rig.
A Complete System
A Planar Lipid Bilayer Workstation provides an integrated environment for measuring transmembrane currents from lipid membranes. This unique apparatus enables the user to perform research using this powerful technology.
A Planar Lipid Bilayer (BLM) workstation, most commonly used to record currents through actively gating, ion conducting, single channels is a complex apparatus requiring several components working in concert. These components include a means to support the lipid membrane, high gain amplification, shielding of electromagnetic and mechanical interference, mechanisms for stirring and changing solutions, signal processing, data analysis, and a means to archive acquired data. A schematic representation of a basic BLM layout is shown above.
A Faraday cage is a conducting enclosure used to shield the sensitive electronics in the headstage from electromagnetic interference generated by noise sources in the vicinity of the apparatus. These sources include exterior lighting, nearby equipment and electrical wiring. The headstage and membrane support system (e.g. cups and chambers) are always housed within the cage. Other devices, such as a perfusion system or stirring apparatus, can be housed within or outside of the cage enclosure. To minimize noise, these components from Warner Instruments are designed to operate comfortably within the cage enclosure.
Faraday cages are commercially available from most table manufacturers and the most common design is that of a copper or aluminum mesh supported on an aluminum frame. Ideally, the cage frame is attached to the leg assembly of a floor-standing vibration isolation table but can also be attached directly to the table top. Entry is through large front panel doors. This design is most often used in conjunction with patch clamp setups since the large enclosure can house a microscope along with other devices.
The isolation and damping of mechanical noise is critical to increasing the signal to noise ratio of a BLM workstation. Several approaches have been employed to eliminate large amplitude mechanical vibrations in an experimental setup and these include specially designed vibration isolation tables or optical benches (both usually floor standing) and an old-school approach of placing heavy sheets on on partially inflated inner tubes or tennis balls.
Floor standing benches employ a massive table top resting on pneumatic supports and we recommend the use of a high quality commercial table since these devices are easy to maintain and provide long term stability. They also provide more effective damping of vibrational noise inputs.
Another, more subtle, source of noise in electrophysiological recording systems is associated with vibration of the headstage. This movement produces a rapidly fluctuating stray capacitance which appears as increased noise in the amplifier output and the effect can be minimized by shock mounting the headstage on a rigid support. The HH-1 headstage holder with magnetic base from Warner Instruments has been developed expressly for this purpose.
The general strategy in the formation of a planar lipid bilayer membrane centers on spanning lipids across a small hole or aperture in a membrane support. A cocktail of lipids, usually suspended in a solvent such as decane, is manually painted or drawn across the aperture. Excess lipids drain away from the aperture and under hydrophobic pressure the remaining lipids order themselves into a molecular bilayer.
Planar lipid bilayer membranes are routinely generated on a variety of supports including cups made from various plastics. Pasteur pipette tips, Teflon or PTFE sheets, or other plastic septa have also been used. These supports are either custom fabricated or are purchased from commercial sources. Currently, the most popular system for supporting artificial bilayer membranes is the cup and chamber design which Warner Instruments manufactures in several combinations of material, volume and aperture size.
The size of the aperture is an important factor in determining the stability of the supported membrane. If the hole diameter is too large, the membrane formed will be electrically noisy and mechanically fragile. Alternatively, a smaller hole diameter reduces capacitive noise and is mechanically more robust. However, the probability of vesicle fusion decreases in a manner inversely proportional to the membrane area. Based on these considerations, its clear that the choice of hole size represents a trade-off between membrane noise, stability, and the probability of channel incorporation. The best hole size and geometry for a particular application is usually determined empirically.
Stirring of solutions in the recording chamber is important for the production of reproducible results, especially following the addition of channel agonists or antagonists. Additionally, stirring facilitates vesicle fusion, presumably by vibrating the bilayer or continually introducing new vesicles to the membrane surface. Ideally, a stirring apparatus should introduce little mechanical and electrical noise to the data stream, particularly during acquisition.
Illumination within a Faraday cage is often an afterthought since its impact is usually one of convenience. However, precise illumination of the aperture in the cup greatly facilitates the ease in which membranes are formed. Ideally, the lamp should introduce no electrical artifacts and the projected light should not heat the materials illuminated. The SUN-1 lamp from Warner Instruments is a bright, halogen, DC light source which remains electrically isolated from the electronics while in use and dissipates greater than 60% of the heat generated out the back of the lamp housing.
Exchanging of solutions (termed perfusion) normally occurs following incorporation of a channel to the bilayer membrane, or when experimental conditions require an alteration in ionic conditions or the removal of previously added compounds.
Ideally, a good perfusion system is capable of exchanging solutions in the recording chamber without interrupting the recording process or rupturing the membrane. The process should also be easy to implement and should proceed rapidly. However, most researchers do not attempt to make recordings while perfusing since this is likely to result in a broken membrane.
Several techniques for solution exchange are available. These include gravity feed devices, pump driven devices, or manually-applied pressure driven systems. Warner Instruments prefers the use of a gravity feed system since this approach introduces the least mechanical noise into the perfusion process. In general, fresh solution is added to the bottom of the recording chamber while perfusate is removed from the top.
A high-quality amplifier is an absolute requirement for recording single channel currents. The amplifier must be capable of resolving currents in the low pA range with very little added noise. Several manufacturers produce amplifiers of high-quality, and the greatest degree of variation between manufacturers has been in headstage design. The simplest headstages use resistive feedback circuitry which allows the amplifier to pass large currents.
Warner Instruments is the only manufacturer to provide an amplifier specifically dedicated to bilayer applications. As such, our BC-535 amplifier has many unique features designed to simplify the user's experience.
Filtering of the amplifier output is essential for resolving discrete channel fluctuations from the large amplitude, high frequency noise present in the signal. Properly applied filtering is important since over-filtering of data will obscure or modify channel gating events (a condition to be avoided!). Our bilayer clamp amplifier provides a built-in 4-pole Bessel filter which can select filtering from 50-20k Hz in 1-2-5 steps, or can be bypassed entirely.
Many researchers opt to filter their data using an external device. This approach provides significantly greater frequency control and the best devices are of the low-pass, 8-pole Bessel design. In general, its better to slightly under-filter data during acquisition since additional filtering can be subsequently applied during analysis. The bilayer Workstation includes the 8-pole LPF-8 Bessel filter for optimal performance.
As indicated earlier, a computer is a necessary component in modern BLM workstations since this device is used both for data acquisition and data analysis. The capabilities of all PCs sold today far exceed the performance requirements of acquisition systems commonly used in bilayer work. (Consult the manufacturer of your acquisition system for specific details.)
As such, the major considerations in the purchase of a computer for the BLM is the hard-disk capacity and data storage method employed. Many educational institutions have purchase agreements with popular computer manufacturers making the acquisition of a computer a simple matter. Please contact our offices if you wish to discuss the best configuration for your application.
Acquisition Hardware and Software
Since the analysis of single channel data is statistical in nature, a large number of channel events are required to produce significant results. This condition naturally lends itself to the use of a computer. However, computers are digital devices and the analog signal from the amplifier must be transformed by an analog to digital (A/D) converter prior to collection.
Most A/D converters are bundled with software which emulates a chart recorder or oscilloscope to aid in data acquisition. In addition, several manufacturers have written software specifically designed to meet the needs of single channel recording. The two most common acquisition packages of this type (acquisition hardware and software) are the pClamp/DigiData suite from Molecular Devices and the PatchMaster/LIH suite from HEKA. Other software packages are available through commercial sources or on the Internet. In addition, many investigators have written their own programs to address their specific needs.
The ability to easily archive and retrieve data is an important component of a BLM workstation. During the course of an average experiment, a significant quantity of data is collected for subsequent analysis.
One advantage of any archival system is that it permits selective access to previously recorded data for subsequent analysis. The choice of the proper system to be used will depend upon the needs of the researcher, the financial resources available, and the type of data acquired (fast or slow channel kinetics resulting in large or small file sizes). We will be glad to discuss these options with you as you consider your computer purchase.
Power Line Conditioning
An often overlooked source of noise in electrophysiological recording is that introduced by fluctuations in the power circuit supplying the apparatus. Many research labs are placed on upper floors in a large research facility where the power circuit is shared by many other labs. Usually, these labs contain large pieces of equipment. NMR's, centrifuges, and refrigerators are notorious for introducing variances in both the power and ground sides of a circuit. While most instrumentation used in BLM work are designed to compensate these variances, their effect cannot be completely abolished in a distributed network of components. Under these conditions the effect of these variances can show up either as a rapidly fluctuating transient or as increased background noise in the data.
An elegant solution to this problem is provided by the use of an isolation transformer to supply power to the entire Workstation. The PowerVar Ground Guard Isolation Transformer is a two-sided device wherein one side connects to the institutionally supplied power and the other, isolated, side connects to the BLM Workstation. This configuration results in the presentation of a stable power source to the BLM Workstation which abolishes noise artifacts introduced from varying loads on the 'house circuit'.
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