Specialized Tools for Electrophysiology and Cell Biology Research

Introduction to the BLM Workstation

Product Summary

The Planar Lipid Bilayer Workstation is an ideal tool for measuring pico- or nano-scale charge currents across a membrane.

  • A complete facility for recording from bilayer membranes
  • Designed to have you quickly up and running
  • Simple, integrated design
  • Popular data acquisition packages available
  • Power line conditioning available
  • On-site setup and training available

This page generally describes the Bilayer Workstation components.
Detailed descriptions can be found here

A Complete System

The Planar Lipid Bilayer Workstation from Warner Instruments integrates every significant component required for the assembly of a working BLM rig. This unique device allows the user to quickly get up to speed in performing research using this powerful technology.

The Design

A Planar Lipid Bilayer (BLM) Workstation , 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.

The Components

O2/CO2 Controllers

Faraday cage

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.

FC Series Faraday cages from Warner Instruments have been specifically designed with the bilayer user in mind. This compact assembly is comprised of a shielded enclosure which completely houses the included vibration isolation table. Warner's table-top design requires relatively little lab space, rests comfortably on a sturdy work surface, and actively isolates the vibration isolation table from the cage enclosure. The cage is easily assembled and has several design features accommodating bilayer work.

Vibration Isolation

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 heavy sheets resting 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. The vibration isolation tables included with FC Series Faraday cages have either active or passive pneumatic supports and equal in performance to the floor standing models commonly used in this application .

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.

Membrane Support

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

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

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.

Perfusion

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.

Signal Amplification

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.

More sophisticated bilayer clamp amplifiers incorporate capacitive feedback circuitry in their headstage design. This modification provides dramatically increased bandwidth and noise performance at the expense of large current passing ability. Since bilayer applications seldom generate currents larger than 200 pA, this trade-off is usually not significant.

Warner Instruments is the only manufacturer to provide an amplifier specifically dedicated to bilayer applications. As such, our amplifier has many unique features designed to simplify the users experience.

Signal Filtering

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.

Computer

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 the basic Pentium class PC commonly sold today far exceeds the performance requirements of acquisition systems commonly used bilayer work. (Consult the manufacturer of your acquisition system for specific details.)

Therefore, the major considerations in the purchase of a computer for the BLM Workstation 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/FitMaster 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.

Data Archival

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 and several devices are commercially available for data storage. These devices include, but are not limited to: VCR tape (requires a signal converter or pulse code modulator), DAT tape, analog tape, portable or removable hard drives, Zip or Jazz drives, or CD-R/CD-RW.

An advantage of these archival systems is that they allow 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.

Oscilloscope/Chart Recorder

While many investigators use software emulated display devices (e.g., a computer) coupled to their acquisition hardware to view data as it is being acquired, others rely on dedicated instrumentation for this purpose. These instruments include chart recorders and oscilloscopes. The primary advantage of an oscilloscope over a chart recorder is one of speed. A chart recorder, however, produces a permanent record that is lacking in an oscilloscope. Software emulation can model one or both of these hardware devices and is both fast and produces a permanent record. Regardless of whether the investigator uses a chart recorder, an oscilloscope, or a software emulated device, the data is previewed during acquisition and is stored for subsequent analysis.

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'.

On-Site Setup and Training

Many investigators first entering the arena of research using the planar lipid bilayer are overwhelmed by the wealth of design and application issues surrounding the proper assembly and use of a Bilayer Workstation. While tractable, this state of affairs can result in an investigator choosing a less effective means to achieve his or her research goals.

Warner Instruments recognizes the need to make this technology more accessible and is the first company to establish on-site assembly and training in the proper care and use of the Bilayer Workstation. Our Director of BLM Development, Dr. Edmond Buck, has 15+ years experience using this powerful technology and is committed to providing extensive support for this important technique.

Dr. Buck will visit your site, assemble the workstation and instruct you in how to use and maintain the equipment. If desired, he can also provide guidance and insight into the best way to use your acquisition and analysis software. (Note: Support and warranty rights reserved by the respective data acquisition manufacturers.)

It is our committed goal to quickly and efficiently optimize your setup and skill set allowing you to focus on data acquisition. We invite you to contact Dr. Buck to discuss your needs and application.

References available

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