Warner Instruments BC-535 User manual

BC-535 Preliminary, Rev. 060126
W
Wa ar rn ne er r I In ns st tr ru um me en nt ts s
B
Bi il la ay ye er r C Cl la am mp p A Am mp pl li if fi ie er r
M
Mo od de el l B BC
C- -5 53 35 5
Warner Instruments
1125 Dixwell Avenue, Hamden, CT 06514
[phone] / [phone]
[phone] - fax

BC-535 Preliminary, Rev. 060126
T
Ta ab bl le e o of f C Co on nt te en nt ts s
NOMENCLATURE....................................................................................................................................5
Text conventions.....................................................................................................................................5
Device panel abbreviations....................................................................................................................5
CONTROL DESCRIPTION......................................................................................................................6
Front panel..............................................................................................................................................6
Hold......................................................................................................................................................6
Offset ....................................................................................................................................................7
Meter ....................................................................................................................................................7
Outputs.................................................................................................................................................8
Capacitance compensation...................................................................................................................8
Power ...................................................................................................................................................9
Rear panel ...............................................................................................................................................9
Headstage.............................................................................................................................................9
Circuit and chassis grounds.................................................................................................................9
Gain Telegraph ..................................................................................................................................10
Filter Telegraph .................................................................................................................................10
Im output ............................................................................................................................................11
External Command In ........................................................................................................................11
Capacitance Output............................................................................................................................11
Cap Sync Out......................................................................................................................................11
External speaker.................................................................................................................................11
ADDITIONAL INFORMATION............................................................................................................11
Headstage connections .........................................................................................................................11
Model membrane..................................................................................................................................12
SETUP........................................................................................................................................................13
Basic design...........................................................................................................................................13
Faraday cage......................................................................................................................................13
Vibration isolation..............................................................................................................................14
Membrane support .............................................................................................................................14
Amplification......................................................................................................................................15
Filtering..............................................................................................................................................15
Acquisition hardware and software ...................................................................................................16
Data analysis......................................................................................................................................16
Data archival......................................................................................................................................16
Stirring ...............................................................................................................................................16

BC-535 Preliminary, Rev. 060126
Perfusion ............................................................................................................................................17
Oscilloscope .......................................................................................................................................17
INITIAL TEST..........................................................................................................................................18
Amplifier setup .....................................................................................................................................18
Overview................................................................................................................................................18
Initial conditions...................................................................................................................................18
Hold voltage test.................................................................................................................................19
Input noise test without model membrane..........................................................................................20
Input noise test with model membrane...............................................................................................20
Test instrument Imoutput....................................................................................................................21
Cap test...............................................................................................................................................21
Autozero .............................................................................................................................................21
Capacity compensation ......................................................................................................................22
OPERATION ............................................................................................................................................23
Setup of the bilayer chamber...............................................................................................................23
Input offset............................................................................................................................................24
Input offset adjustment .......................................................................................................................24
Bilayer formation..................................................................................................................................24
Commands.............................................................................................................................................25
APPENDIX................................................................................................................................................26
Theoretical considerations...................................................................................................................26
Shielding.............................................................................................................................................26
Grounding ..........................................................................................................................................26
Membrane capacitance calculations...................................................................................................28
Suggested References ...........................................................................................................................29
Specifications.........................................................................................................................................30
Chloriding electrodes ...........................................................................................................................32
Techniques for chloriding silver wires................................................................................................32
Accessories and replacement parts.....................................................................................................33
Warranty...............................................................................................................................................33
Service....................................................................................................................................................33
Service notes.......................................................................................................................................33
Certifications.........................................................................................................................................35
Glossary.................................................................................................................................................38

BC-535 Preliminary, Rev. 060126
The Warner BC-535 Bilayer Clamp Amplifier is a resistive-feedback voltage clamp amplifier
designed specifically for applications using planar lipid bilayer membranes. The unique circuitry and
dedicated design of this amplifier allows Warner to present an instrument of broad capability and
superior quality at a cost significantly below that of our competitors.
The operational range of the BC-535 has been enhanced by the introduction of dual feedback-
resistor circuitry within the headstage. This enhancement allows the amplifier to comfortably pass
currents of up to 2 nA while preserving the sub-pA sensitivity of the instrument. In addition, the range
of the digital hold control has been extended to 400 mV for internally generated commands and the
amplifier supports up to 1 V at the external command input, for a sum capability of 1400 mV hold
potential.
The remaining functionality of the BC-353 is built on the renown capabilities of the BC-525D and
includes junction potential auto-zeroing, a unique multi-step, digital hold potential circuit, audio
monitoring of membrane formation, and direct readout of the membrane capacitance.
Features of the BC-353 include
9Dedicated design for bilayer applications
9Digital, multi-step hold potential control
9Hold potentials to ±1400 mV
9Currents to ±2000 pA
9Input offset with Auto-Zero
9Direct membrane capacitance measurement
9Low-pass 4-pole Bessel filter
9Audio output
9Capacitance compensation circuitry
THIS EQUIPMENT IS NOT DESIGNED NOR INTENDED
FOR USE ON HUMAN SUBJECTS

BC-535 Preliminary, Rev. 060126 5
NOMENCLATURE
Text conventions
This manual refers to amplifier controls at three functional levels; control blocks, specific controls
within a block, and settings of specific controls. To reduce confusion, we have employed several text
conventions which are specified below. Since our goal is to provide clarity rather than complexity, we
welcome any feedback you may wish to provide.
¾Warner Instrument product numbers are presented using bold type.
¾References to instrument panel control blocks are specified using UNDERLINED SMALL CAPS.
¾References to specific controls within a block are specified using NON-UNDERLINED SMALL CAPS.
¾Finally, references to individual control settings are specified in italic type.
¾Special comments and warnings are presented in highlighted text.
Any other formatting should be apparent from context.
Device panel abbreviations
The BC-353 has several abbreviations on the front panel. They are listed here for quick
reference. In addition, these and other terms are collected and included in a Glossary at the back of
this manual.
Term Meaning Section
Vccommand voltage METER,OUTPUTS
Imoutput current METER,OUTPUTS
CMD IN commend in
CAP TEST capacitance test METER
CAP COMP capacitance compensation CAP COMP
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BC-535 Preliminary, Rev. 060126 6
CONTROL DESCRIPTION
The instrument front panel is divided into six control blocks titled HOLD, OFFSET, METER,OUTPUTS,
CAP COMP, and POWER. The instrument rear panel has BNC connectors for the GAIN and FILTER
TELEGRAPHS, IMOUTPUT, CAP SYNC, MEMBRANE CAPACITANCE, and EXTERNAL COMMAND IN. A 9-pin DIN
connector (for the headstage), a 15 pin D connector, binding posts for CIRCUIT and CHASSIS GROUND,
and a SPEAKER OUTPUT are also located on the rear panel.
Front panel
Hold
The HOLD block contains a meter and
controls for the application of internal or
external VmHOLD commands.
The appropriate membrane holding
potential is achieved by summing the
selected HOLD voltages (internal plus
external) with the INPUT OFFSET voltage
which results in a corrected transmembrane
voltage. An LED indicates COMMANDS
APPLIED to the headstage.
The internal HOLD control is comprised
of a digital circuit providing discrete
adjustment of the command potential. Two toggle switches directly below the COMMANDS APPLIED
meter are used to step the applied command by ± 10 or ± 1 mV, respectively. A black push button is
used to quickly swap the polarity of the applied holding potential. The maximum range for this control
is
± 400 mV.
The internally generated HOLD command can be disabled by selecting the off position on the
ON/OFF TOGGLE SWITCH to the right of the meter.
Note: The METER will still display the programmed hold voltage when the ON/OFF TOGGLE SWITCH is
selected to off. However, the programmed command will not be applied and the COMMANDS APPLIED
LED will remain unlit. This feature allows the user to select or change the holding potential without
applying it to the membrane.
The Vcx 10 OUTPUT monitors the voltage command applied to the headstage multiplied by 10.
This output reports the sum of potentials from VmHOLD, CMD IN, and PULSE GENERATOR. Connection is
made via BNC’s located on both the front and rear panels of the amplifier.
The ImOUTPUT reports the membrane current modified by amplifier gain and/or internal filtering.
Imoutput BNC’s are located on both the front and rear panels of the instrument.
External commands are applied to the amplifier via the COMMAND INPUT BNC connectors located
on both the front and rear panels. The FRONT/REAR TOGGLE SWITCH either disables all external input or
selects the location for command inputs. Selectable attenuation values are x0.1, x0.01, or x0.001.
Externally generated COMMAND INPUTS are summed with the internally generated HOLD voltage.
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BC-535 Preliminary, Rev. 060126 7
Offset
The OFFSET block contains the INPUT OFFSET and the AUTO-ZERO
controls.
The INPUT OFFSET section is comprised of a rotary potentiometer with
low/high LED’s, the AUTO-ZERO pushbutton, the UNLOCK toggle, and an
ACTIVE LED. This section is used to compensate for junction potentials
produced by dissimilar solutions or other electrode potential differences.
The OFFSET circuit (AUTO-ZERO and OFFSET CONTROL) must be armed
prior to use. This is achieved by use of the UNLOCK TOGGLE. This toggle is
of the momentary-on style and is operated by an upward movement.
When the circuit is armed the ACTIVE LED will be lit. Offset adjustments
can then be easily achieved by use of the AUTO-ZERO control or ROTARY
POTENTIOMETER. The circuit can be disarmed by a second movement of the UNLOCK TOGGLE.
The AUTO-ZERO control provides the most direct means for setting the junction potential. When
armed, pressing the pushbutton initiates a cycle wherein the amplifier searches for and sets the offset
potential. The offset circuit is automatically disarmed at the completion of the cycle. Cycle time is
approximately 1 s.
The ROTARY POTENTIOMETER is used to provide manual adjustment of up to ±120 mV at the
headstage input. Manual adjustment is only available when the offset circuit is armed. Fine adjustment
of the rotary control can be achieved by pressing the control in while turning. Low/high LED’s are
provided to indicate which direction the manual offset control should be adjusted to achieve a null
junction potential setting. The offset circuit must be manually disarmed when using this control.
In both cases, the applied OFFSET potential can be monitored on the METER by selecting OFFSET in
the METER block.
Meter
The METER block contains a 3.5 digit LED METER and a four
position switch for selecting OFFSET, CAP TEST, current output (Im), or
voltage command (ΣVc).
Selection of OFFSET displays the potential applied to the
headstage via the MANUAL or AUTO-ZERO control located in the OFFSET
block. Offset potential is displayed in units of pA. Alternatively, this
display indicates the potential required to bring Imto zero when the
command input is set to zero.
Selection of CAP TEST places the instrument into capacitance test
mode. This useful mode dynamically tests and reports the membrane
capacitance. Capacitance values are reported on the meter in units of
pF. A rear panel BNC also reports the calculated membrane capacitance whenever CAP TEST is
selected. Reported units are 1 mV/pF.
Selection of Imdisplays the value of the DC current presented at the ImOUTPUT BNC. The meter is
capable of displaying currents up to ±1999 pA.
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A Harvard Apparatus Company .

BC-535 Preliminary, Rev. 060126 8
Selection of ΣVcdisplays the sum of all command voltages (VmHOLD and COMMAND INPUT) applied
to the headstage. The meter is capable of displaying command voltages up to ±1999 mV. The meter
displays DC values and will average AC signals or pulses.
Outputs
The OUTPUTS block contains controls for
selecting the ImGAIN of the amplifier and signal
filtering using the built-in 4-pole Bessel filter. This
block also contains the audio output controls.
Amplifier gain is selected via an 11 position
selector switch. Gain settings are from 0.5 to
1000 mV/pA in 1-2-5 steps. For transmission to
external devices, the selected gain setting appears
as a defined voltage at the GAIN TELEGRAPH BNC
on the instrument rear panel.
Internal filtering of the Imsignal is selected
via a 9 position selector switch. Filter settings are
from 50 Hz to 20 kHz in 1-2-5 steps. A BYPASS
TOGGLE switch bypasses the 4-pole Bessel filter and presents the full bandwidth (75 kHz) of the
amplifier at the ImOUTPUT. Filtering is applied post-gain. For transmission to external devices, the
selected filter setting appears as a defined voltage at the FILTER TELEGRAPH BNC on the instrument
rear panel.
The AUDIO section is comprised of an on/off toggle and volume control. Audio output is useful
during membrane formation to monitor the successful application of lipids. An open hole prior to
membrane formation produces a characteristic low frequency sound while the same aperture with
membrane produces a different characteristically higher frequency sound. The pitch of the signal is
keyed to the membrane capacitance and will increase as the capacitance increases allowing non-visual
monitoring of membrane ‘thinning’.
Capacitance compensation
The capacitance compensation circuit allows for cancellation
of large currents (capacity currents) generated when a step
potential is applied to the bilayer membrane.
The CAP COMP block contains controls for the adjustment of
AMPLITUDE and TIME CONSTANT for both FAST (0-10 µs) and SLOW
(0-10 ms) components of the current. The adjustment is made
in pairs, that is, the FAST pair (AMPLITUDE and TIME CONSTANT) is
first adjusted to minimize the transient, followed by adjustment
of the SLOW pair. Each pair is adjusted in turn as many times as
required to completely minimize the transient.
The AMPLITUDE control for the FAST component is a ten turn
potentiometer with a counting dial and can be used to provide a
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BC-535 Preliminary, Rev. 060126 9
reading of the capacitance in pF. The dial is calibrated to 50 pF/turn.
Power
Immediately adjacent to the CAP COMP block is the master power switch for the
BC-535. An LED indicates power on status.
Rear panel
The instrument rear panel has BNC connectors for GAIN and FILTER TELEGRAPHS, IM
OUTPUT, CAP SYNC, EXTERNAL RESET IN, and EXTERNAL COMMAND IN. A 9-pin DIN
connector (for the headstage), a 15 pin I/O INTERFACE, binding posts for CIRCUIT and
CHASSIS GROUND,and a SPEAKER OUTPUT are also located on the rear panel.
The photo below shows the various attachment points on the instrument rear panel. Connections
are described right-to-left.
Headstage
The HEADSTAGE is housed in a small aluminum enclosure and connects to the amplifier via a 1.8 meter
cable. A 9-pin DIN connector is provided for this attachment.
Note: When routing the headstage cable from your Faraday cage to the instrument, we recommend
intertwining the headstage and ground cables to minimize ground loops.
Circuit and chassis grounds
CIRCUIT and CHASSIS GROUND binding posts are provided at the rear of the amplifier to allow
modification of instrument grounding.
The CHASSIS GROUND binding post is internally connected to the green-wire ground of the power
plug. Therefore the instrument does not normally require a separate ground. However, it may become
necessary to independently ground the chassis of the BC-535 when it is used as a freestanding device
and not incorporated into a rack.
The CIRCUIT GROUND binding post allows external connection to the internal ground circuitry of
the amplifier. This post is used to provide a common circuit ground point for all active components
(Faraday cage and contents, SUNStir-3 assembly, temperature controller, etc.) within the bilayer rig,
thus preventing ground loops.
In general, the internal circuitry of the BC-535 maintains a virtual ground and is not normally not
connected to chassis ground. However, when necessary, the CIRCUIT GROUND binding post can be used
to tie the circuit and chassis grounds to a common potential. This is the default configuration when the
instrument is shipped from the factory.
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BC-535 Preliminary, Rev. 060126 10
Note: We recommend separating the circuit and chassis grounds by disconnecting the bridging bar
between the associated ground posts. Loosen the posts and slide the bridge to one side.
Gain Telegraph
The GAIN TELEGRAPH is a stepped voltage output designed to communicate the instrument gain
setting to your acquisition software. DC voltages are stepped from 0.0 V to 5.5 V, in steps of 500 mV.
GAIN TELEGRAPH voltage outputs for the associated amplifier ImGAIN are specified below. (ImGAIN
settings are selectable in the front panel OUTPUTS block, see page 8)
ImGain (mV/pA) Gain Telegraph (V)
standby 0.0
0.5 0.5
1 1.0
2 1.5
5 2.0
10 2.5
20 3.0
50 3.5
100 4.0
200 4.5
500 5.0
1000 5.5
Filter Telegraph
The FILTER TELEGRAPH is a stepped voltage output designed to communicate the instrument filter
cutoff frequency setting to your acquisition software. DC voltages are stepped from 0.5 V to 5.0 V, in
steps of 500 mV and are specified below. (FILTER settings are selectable in the front panel OUTPUTS
block, see page 8)
Filter Frequency (Hz) Filter Telegraph (V)
50 0.5
100 1.0
200 1.5
500 2.0
1k 2.5
2k 3.0
5k 3.5
10k 4.0
20k 4.5
bypass 5.0
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BC-535 Preliminary, Rev. 060126 11
Im output
The ImOUTPUT signal present on the instrument front panel is mirrored on this rear panel BNC.
Use of this output rather than the front panel BNC can unclutter your work environment.
External Command In
External COMMAND IN signals can be input via BNC connectors on either the instrument front
panel or the instrument rear panel. Input location is selectable by the front/rear COMMAND INPUT
toggle switch located in the HOLD control block on the instrument front panel. Use of the rear input
can unclutter your work environment.
Capacitance Output
The calculated membrane capacitance is output on this BNC when the instrument is in CAP TEST
mode. Switching the METER selector switch to CAP TEST activates the CAP TEST circuit. This feature is
useful for recording the calculated membrane capacitance into a chart recorder or data acquisition
system. Capacitance output values are 1 mV/pF.
Cap Sync Out
This signal is used to synchronize an oscilloscope or other device with the BC-535 when using the
CAP TEST function. The SYNC OUT signal is keyed to the peak of the triangular wave for CAP TEST which
corresponds to the leading edge of the resulting square wave. The SYNC OUT signal is a standard TTL
square wave and is 100 µs in duration.
External speaker
A standard ¼” RCA jack is provided for attachment to an external speaker for use in
environments where the ambient noise exceeds the volume capabilities of the internal speaker.
ADDITIONAL INFORMATION
Headstage connections
The HEADSTAGE is housed in a small aluminum enclosure and connects to the amplifier via a 1.8
meter cable. Electrode connections are made to two 1 mm mini-jacks marked INPUT and REF
(reference). A third mini-jack (GND; circuit ground) is located on the side of the headstage for
connecting to shields or grounding equipment.
The ground connection on the headstage merits specific discussion. The headstage case is
internally connected to the command potential (INPUT electrode) of the headstage. As a result, the
headstage does not require a separate ground. However, the isolated grounding jack on the headstage
is provided as a means to ground a small Faraday cage through the headstage if the user desires.
Notes:
1. If the Faraday cage is grounded through the headstage (not recommended), then do not run
a separate ground connection from the Faraday cage to any other ground point.
2. Do not connect the ground on the headstage to either the input or ref electrode as this will
disable the amplifier.
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A Harvard Apparatus Company .

BC-535 Preliminary, Rev. 060126 12
Model membrane
The BC-535 is shipped with a model membrane, the MC-1, which can be used to test the
performance of the amplifier. The MC-1 contains a 100 pF capacitor connected in parallel with a 1 GΩ
resistor. The precision of this resistor is ± 5%.
The MC-1 connects to the two 1 mm mini-jacks on the headstage marked INPUT and REF. The
green grounding wire on the MC-1 is attached to the isolated grounding jack on the headstage.
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A Harvard Apparatus Company .

BC-535 Preliminary, Rev. 060126 13
SETUP
For those with little experience in bilayer work, we suggest a review of Ion Channel
Reconstitution edited by C. Miller, Plenum Press, New York, 1986. In particular, Chapter 5, "How To
Set Up A Bilayer System", covers many important aspects of the subject. Several other pertinent
references are included in the appendix at the back of this manual.
Figure 1. Schematic representation of a BLM setup.
Basic 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 interference, shielding of mechanical vibration, mechanisms for stirring and
changing solutions, signal filtering, data acquisition analysis, and a means to archive acquired data.
A schematic representation of a basic BLM layout is shown in Figure 1. Warner Instruments
provides all components used in the assembly of a BLM workstation, including Faraday cages,
vibration isolation tables, a dedicated bilayer clamp amplifier, high quality signal filtering devices,
illumination and stirring mechanisms, cups and chambers, and perfusion apparatus.
The components listed above may be assembled in various ways to achieve a working system.
Regardless of the configuration used, care must be taken in the design of a BLM workstation to
minimize both mechanical and electrical noise sources since single channel currents are often only a
few pA in magnitude. In this section we describe the basic design of a BLM workstation.
Faraday cage
A Faraday cage is an enclosure designed 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 instrumentation and electrical wiring. The cage can be
fabricated from any conducting material and is grounded. While the design of the BC-535 facilitates
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BC-535 Preliminary, Rev. 060126 14
grounding the Faraday cage through the headstage (see Headstage connections, page 11), we do not
recommend this procedure, but instead suggest that the cage be grounded through the amplifier
circuit ground.
Several Faraday cage designs are available. The most common commercial design is that of a
copper or aluminum wire mesh supported on an aluminum frame. This frame attaches to the
conducting top of a floor-standing vibration isolation table which completes the cage enclosure. 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 as well as several other devices.
An option exclusively presented by Warner Instruments consists of a Faraday cage with an
enclosed vibration isolation table. This unique combination is specifically designed with the bilayer user
in mind. The assembly requires little lab space, rests comfortably on a sturdy work surface, and
actively isolates the tabletop from the cage enclosure. The cage is easily assembled and has several
design features simplifying bilayer work.
Regardless of the Faraday cage employed, the headstage and membrane support system (e.g.
cups and chambers) are contained within the cage which acts as the electromagnetic shield. Other
devices such as a perfusion system or stirring apparatus may also be housed within the cage, but
some investigators place these components on the outside (with proper grounding) to reduce their
noise contribution.
Vibration isolation
The isolation and damping of mechanical noise is critical to increasing the signal to noise ratio of
a BLM workstation. The significance of this becomes apparent when one considers that the acoustic
coupling of normal speech to the buffers on each side of the bilayer is large enough to present a
significant capacitance current artifact in the data.
Several approaches have been employed to eliminate large amplitude mechanical vibrations in an
experimental setup. These include specially designed vibration isolation tables or optical benches.
These floor standing benches employ a heavy table top resting on pneumatic supports. Alternatively,
investigators have placed heavy concrete slabs (commonly referred to as balance tables) or large steel
sheets on partially inflated inner tubes or tennis balls. We recommend the use of a high quality
commercial table since these devices provide more long term stability and more effectively damp
vibrational noise inputs.
Another, more subtle, source of noise in electrophysiological recording systems is associated with
vibration of the headstage. This movement can produce a rapidly fluctuating stray capacitance which
appears as increased noise in the amplifier output. This effect can be minimized by shock mounting
the headstage to its support. Since it is advantageous to keep the associated moment arm as small as
possible, the headstage should be directly mounted to its support rather than through a long
connecting rod. Warner Instruments has developed the HST-1 headstage holder system expressly for
this purpose.
Membrane support
The general approach to the formation of a planar lipid bilayer membrane involves spanning lipids
across a small hole or aperture in a membrane support. A cocktail of lipids, usually suspended in a
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BC-535 Preliminary, Rev. 060126 15
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 orient themselves into a
molecular bilayer.
Planar lipid bilayer membranes are routinely generated on a variety of supports including cups
made from polystyrene, polysulfone, Teflon, or Delrin. Teflon sheets, Pasteur pipette tips, or plastic
septa have also been used. These supports are either custom fabricated for the desired application or
are purchased from commercial sources. Currently, the most commonly used system for supporting
artificial bilayer membranes is the cup/chamber design. Warner Instruments manufactures cups and
chambers in several combinations of material, cup volume and aperture size. These may be viewed in
our catalog or at our website under the model numbers BCH-13,BCH-22 and BCH-P.
The geometry of the aperture is important to the stability of the supported membrane. If the hole
diameter is too large then the membrane formed will be electrically noisy and mechanically fragile. A
smaller hole diameter reduces electrical noise and is mechanically more robust, however, the
probability that a vesicle will fuse to the membrane is inversely proportional to the membrane size.
The simplest aperture geometry is that of a tubular channel drilled through the supporting septa.
This geometry has the advantage of being easy to manufacture and maintain. It is generally assumed
that the membrane formed is maintained at one end of the bore. This is the design employed by
Warner cups.
Another aperture geometry commonly used is that of a conical hole with the small end of the hole
supporting the bilayer membrane. This geometry, often employed on custom made cups, is generally
formed by melting a small bubble (or boss) in the cup wall from the inside using a heated piece of
pointed metal, and then shaving the outside boss away with a razor until a hole of the desired
diameter is achieved.
Based on the above discussion, it is clear that the choice of hole size and geometry represents a
trade-off between membrane noise, fragility, and the probability of vesicle fusion. The best hole for a
particular application is usually determined empirically.
Amplification
A high-quality amplifier is an absolute requirement for recording single channel currents. The
amplifier must be capable of resolving currents as low as 1 pA with very little added noise. While
several manufacturers today produce amplifiers of high-quality, the greatest degree of variation
between instruments is in the feature set. Warner Instruments is the only manufacturer to produce a
dedicated bilayer clamp amplifier, and it’s performance and feature set have been optimized for
bilayer work.
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 the data will obscure or modify channel gating events (a condition to be avoided!),
while an under-filtered signal will not clearly resolve single channel events. The BC-535 provides a
built-in 4-pole Bessel filter which can be used to select filtering from 50 Hz to 20 kHz, or can be
bypassed.
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BC-535 Preliminary, Rev. 060126 16
Optionally, many researchers filter their data using an external device. These devices are
normally of the low-pass 8-pole Bessel design. While Butterworth filters have steeper frequency cutoff
characteristics, they are less commonly used since they tend to overshooting a rapidly varying signal
thus introducing an artifact into the data. In general, it is better to slightly under-filter the data being
acquired in real-time since additional filtering can be performed later in the analysis software.
Warner Instruments provides a number of filtering devices which can be used in conjunction with
the BC-535 to achieve a high degree of filter resolution. We recommend the use of a high quality
8-pole Bessel filter such as the LPF-8.
Acquisition hardware and software
Since the analysis of single channel data is statistical in nature, a large number of channel events
is required to produce significant results. This condition naturally lends itself to the use of a computer.
However, since computers function digitally, the analog signal from the amplifier must first be
digitized by an analog to digital (A/D) converter prior to analysis. Many A/D converters are bundled
with software which emulates a chart recorder or oscilloscope to aid in data acquisition.
Since single channel gating kinetics can range from sub-ms open times to gating transitions
lasting several seconds or more, the desired characteristics of a high-quality A/D converter include
rapid response times, high signal resolution and low noise.
Data analysis
Once the data has been acquired and stored, it must be analyzed for its biophysical
characteristics. Since the volume of data collected is often exceedingly large, analysis is usually
performed by dedicated software programs. The single most popular program for this purpose is
pClamp (Molecular Devices, Sunnyvale, CA). However, several competing software packages are
available commercially 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 large volume of data is collected for later analysis.
Several devices are available for data storage. These devices include, but are not limited to: VCR tape
(requires a signal converter or pulse code modulator), DAT tape, portable or removable hard drives,
Zip or Jazz drives, CD-RW, or the newer DVD-RW technology.
An important advantage of most of these archival systems is that they allow selective access to
previously recorded data for subsequent analysis. The choice of the proper system 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).
Stirring
Stirring of solutions in the recording chamber is important for the production of reproducible
results, especially following the addition of agonists or antagonists. Additionally, stirring is thought to
facilitate the fusion of vesicles to the bilayer membrane, presumably by vibrating the membrane or by
continuously introducing new vesicles to the bilayer. Ideally, the stirring process should produce
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BC-535 Preliminary, Rev. 060126 17
sufficiently little mechanical noise such that one is able to make recordings while simultaneously
stirring.
Warner Instruments has developed a unique stirplate specifically designed for the planar lipid
bilayer. This stirplate, the SPIN-2, provides separate rotating dipoles for each side (cis and trans) of
the cup/chamber and its noise-free operation allows data recording while stirring.
Perfusion
Exchange of solutions (termed perfusion) normally occurs following incorporation of a channel to
the bilayer membrane, when experimental conditions require an alteration in ionic conditions, or to
remove a previously added compound.
Under ideal circumstances a good perfusion system is capable of exchanging solutions in the
recording chamber without interrupting the recording process or rupturing the membrane. 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, pump driven
devices, or manually-applied pressure driven systems. In general, fresh solution is added to the
bottom of the recording chamber while the perfusate is removed from the top. Warner Instruments
has developed the BPS-2, an easily assembled ‘traditional’ perfusion system, which integrates well
with our Bilayer Workstation.
Oscilloscope
While many investigators use software emulated display devices coupled to their acquisition
hardware to view data during acquisition, others rely on dedicated instrumentation for this purpose.
These dedicated 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 either of these hardware devices. 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.
Warner Instruments
A Harvard Apparatus Company .

BC-535 Preliminary, Rev. 060126 18
INITIAL TEST
This section describes the basic setup for incorporating the amplifier and headstage into the BLM
workstation. Procedures for testing the performance of the BC-535 immediately follow.
Amplifier setup
The headstage connects to the amplifier via a 1.8 meter cable. It is a good idea to route the cable
through a short shield (anti-wave guide) prior to its entering the Faraday cage. The presence of the
shield will have no effect on the signal received by the amplifier but can help reduce the amount of
electromagnetic interference entering the Faraday cage through the opening.
Since movement of the headstage can appear as a fluctuating stray capacitance at the headstage
input, the headstage should be rigidly mounted to a fixed support. The headstage is shipped mounted
to a platform that can be attached to various holders or micro-manipulators. In addition, the
headstage should be placed as close to the preparation as possible to reduce noise due to increased
input capacitance.
Overview
The procedures described here are provided to verify the functionality of the BC-535. These
procedures should be performed when you first acquire the amplifier and can also be used re-assess
the performance of the amplifier at a later time.
To perform these tests you will need:
•BC-535 Bilayer Clamp amplifier
•included headstage
•included model membrane
•Faraday cage
•an oscilloscope (storage scope if available)
•2 BNC connector cables
Initial conditions
Verify that the amplifier is disconnected and the power switch is off.
Place the HEADSTAGE into the Faraday cage. If using a small (1 cu ft or less) Faraday cage, then
connect the cage to the GROUNDING JACK (green plug)on the HEADSTAGE. If using a larger Faraday
cage, then make a ground connection from the cage enclosure to the coupled CHASSIS/CIRCUIT
GROUNDS on the rear of the BC-535.
Connect the headstage to the amplifier via the input on the rear panel. Connect the provided
power cable from the amplifier to your line source. You are now ready to begin the functional check of
the BC-535.
CAUTION: Connection of the BC-535 to the wrong line voltage could result in severe damage to
the instrument. Therefore, before connecting the amplifier to the power source, check the serial
number label on the rear of the amplifier for its voltage rating. Prior to turning on the BC-535 for the
first time, verify that the line voltage is correct for the instrument. If the instrument voltage rating is
incorrect for your area, contact our Service Department.
Warner Instruments
A Harvard Apparatus Company .

BC-535 Preliminary, Rev. 060126 19
Set all controls on the instrument to the values specified below.
Control block Control Setting
HOLD OPERATE TOGGLE standby
COMMANDS APPLIED TOGGLE off
VOLTAGE UP/DOWN TOGGLES adjust until meter reads 0
COMMAND INPUT FRONT/REAR
TOGGLE off
COMMAND INPUT ATTENUATION
TOGGLE x0.01
OFFSET AUTOZERO inactive
(ACTIVE LED is off)
METER SELECTOR SWITCH
Σ Vc
OUTPUTS GAIN SELECTOR 10 mV/pA
FILTER SELECTOR 5 kHz
FILTER TOGGLE active
AUDIO TOGGLE off
CAP COMP ALL CONTROLS Set to zero
Set the oscilloscope to the following settings:
•Connect Vcx10 OUTPUT to the input of
your oscilloscope.
•time base to 5 ms/div
•voltage base to 0.2 V/div
•auto trigger
•DC coupling on the input channel
Turn on both the BC-535 and the oscilloscope.
Hold voltage test
•Connect the model membrane (MC-1) to the headstage inputs and the green wire from the
model membrane to the headstage grounding jack. Insure that the Faraday cage remains
grounded, either through the headstage grounding jack or to the amplifier circuit grounding
post.
•Switch the STANDBY/OPERATE switch to operate.
•Switch the SELECTOR SWITCH in the METER block to offset.
•Activate the OFFSET CONTROLS in the OFFSET block by moving the UNLOCK toggle to the unlock
position. The ACTIVE LED will light and the toggle switch will return to its original position.
•Adjust the MANUAL INPUT OFFSET control (the twiddle-knob under the LOW/HIGH LEDS in the
OFFSET block) until the meter in the METER block reads 0 mV.
Warner Instruments
A Harvard Apparatus Company .

BC-535 Preliminary, Rev. 060126 20
•Deactivate the OFFSET CONTROLS by moving the UNLOCK toggle to the unlock position. The
ACTIVE LED will go off and the toggle will return to its original position. The offset reading on
the METER in the METER block will not change.
•Adjust the position of the trace on the oscilloscope to a convenient reference line.
•Switch the SELECTOR SWITCH in the METER block to
Σ Vc.
•Using the VOLTAGE UP/DOWN TOGGLE switches in the HOLD block, adjust hold potential until
the meter reads 20 mV.
•Switch the COMMANDS APPLIED toggle in the HOLD block to on. The COMMANDS APPLIED LED will
light.
•Notice that the displayed voltage on the oscilloscope moves from zero to 0.2 V (one division
upward).
•Notice that the METER also shows 20 mV. This reading is correct since the VcX 10 OUTPUT is the
applied VmHOLD multiplied by 10 (e.g.: 20 mV x 10 = 0.2 V).
•Switch the amplifier to standby. Notice that the applied voltage appears at the headstage
input even when the amplifier is in standby mode or when the headstage has open inputs
(i.e. the oscilloscope trace doesn’t change when amp is in standby).
•Make similar adjustments to the hold control to assure yourself that the amplifier performs as
expected.
•Switch the COMMANDS APPLIED toggle in the HOLD block to off.
•Using the VOLTAGE UP/DOWN TOGGLE switches in the HOLD block, adjust hold potential until
the meter reads 0 mV.
•Place the amplifier into standby.
Input noise test without model membrane
•Remove the model membrane. Insure that the Faraday cage remains grounded, either
through the headstage grounding jack or to the amplifier grounding posts.
•Move the BNC connection from the VmX 10 OUTPUT to the ImOUTPUT (in the OUTPUTS block)
and monitor the ImOUTPUT with the oscilloscope.
•Set the amplifier GAIN to 100 mV/pA and the oscilloscope voltage base to 50 mV/div.
•Verify that the FILTER TOGGLE is active and switch the FILTER control to 1 kHz.
•Verify that the COMMANDS APPLIED toggle in the HOLD block is set to off.
•Switch the STANDBY/OPERATE switch to operate. You should observe that the noise level
decreases to no greater than 30 mV p-p. At a gain setting of 100 mV/pA this corresponds to a
maximum noise level of 0.043 pA RMS.
•Place the amplifier into standby.
Input noise test with model membrane
•Connect the model membrane (MC-1) to the headstage inputs and the green wire from the
model membrane to the headstage grounding jack. Insure that the Faraday cage remains
grounded, either through the headstage grounding jack or to the amplifier grounding posts.
•Set the ImGAIN on the amplifier to 5 mV/pA.
•Set the oscilloscope voltage base to 10 mV/div.
•Verify that the filter is set to 1 kHz.
•Verify that the COMMANDS APPLIED toggle in the HOLD block is set to off.
Warner Instruments
A Harvard Apparatus Company .