Warner Instruments BC-535 User Manual
BC-535 Preliminary, Rev. 060126
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Warner Instruments
1125 Dixwell Avenue, Hamden, CT 06514
(800) 599-4203 / (203) 776-0664
(203) 776-1278 - fax
BC-535 Preliminary, Rev. 060126
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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 Im output....................................................................................................................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 feedbackresistor 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
9 Dedicated design for bilayer applications
9 Digital, multi-step hold potential control
9 Hold potentials to ±1400 mV
9 Currents to ±2000 pA
9 Input offset with Auto-Zero
9 Direct membrane capacitance measurement
9 Low-pass 4-pole Bessel filter
9 Audio output
9 Capacitance 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.
¾ Warner Instrument product numbers are presented using
¾ References to instrument panel control blocks are specified using
¾ References to specific controls within a block are specified using
bold type.
UNDERLINED SMALL CAPS.
NON-UNDERLINED SMALL CAPS.
¾ Finally, references to individual control settings are specified in italic type.
¾ Special 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 incl uded in a Glossary at the back of
this manual.
Term Meaning Section
Vc command voltage METER, OUTPUTS
Im output 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
, I
TELEGRAPHS
connector (for the headstage), a 15 pin D connector, binding posts for
OUTPUT, CAP SYNC, MEMBRANE CAPACITANCE, and EXTERNAL COMMAND IN. A 9-pin DIN
M
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 V
The appropriate membrane holding
potential is achieved by summing the
selected
external) with the
which results in a corrected transmembrane
voltage. An LED indicates
APPLIED to the headstage.
HOLD commands.
m
HOLD voltages (internal plus
INPUT OFFSET voltage
COMMANDS
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
±
400 mV.
is
The internally generated
ON/OFF TOGGLE SWITCH to the right of the meter.
HOLD command can be disabled by selecting the off position on the
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 wi thout
applying it to the membrane.
The V
This output reports the sum of potentials from V
x 10 OUTPUT monitors the voltage command applied to the headstage mul tiplied by 10.
c
HOLD, CMD IN, and PULSE GENERATOR. Connection is
m
made via BNC’s located on both the front and rear panels of the amplifier.
The I
output BNC’s are located on both the front and rear panels of the instrument.
I
m
External commands are applied to the amplifier via the
on both the front and rear panels. The
OUTPUT reports the membrane current modified by ampli fier gain and/or internal filtering.
m
COMMAND INPUT BNC connectors located
FRONT/REAR TOGGLE SWIT CH 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
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. Thi s is achieved by use of the
of the momentary-on style and is operated by an upward movement.
When the circuit is armed the
can then be easily achieved by use of the
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.
AUTO-ZERO pushbutton, the UNLOCK toggle, and an
UNLOCK TOGGLE. This toggle is
ACTIVE LED will be lit. Offset adjustments
AUTO-ZERO control or ROTARY
ROTARY POTENTIOMETER is used to provide manual adjustment of up to ±120 mV at the
The
headstage input. Manual adjustment is only available when the offset circuit is armed.
of the rotary control can be achieved by pressing the control in while turning.
provided to indicate which direction the manual
offset control should be adjusted to achieve a null
Fine adjustment
Low/high LED’s are
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
METER block.
the
Meter
The METER block contains a 3.5 digit LED METER and a four
position switch for selecting
voltage command (ΣV
Selection of
headstage via the
c
OFFSET displays the potential applied to the
MANUAL or AUTO-ZERO control located in the OFFSET
OFFSET, CAP TEST, current output (I
).
block. Offset potential is displayed in units of pA. Alternatively, this
display indicates the potential required to bring I
to zero when the
m
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
selected. Reported units are 1 mV/pF.
), or
m
CAP TEST is
Selection of Im displays the value of the DC current presented at the Im OUTPUT BNC. The meter is
capable of displaying currents up to ±1999 pA.
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Instruments
BC-535 Preliminary, Rev. 060126 8
Selection of ΣVc displays the sum of all command voltages (Vm HOLD 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 I
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
on the instrument rear panel.
Internal filtering of the Im signal is selected
via a 9 position selector switch. Filter settings are
from 50 Hz to 20 kHz in 1-2-5 steps. A
TOGGLE switch bypasses the 4-pole Bessel filter and presents the full bandwidth (75 kHz) of the
amplifier at the I
selected filter setting appears as a defined voltage at the
rear panel.
GAIN of the amplifier and signal
m
GAIN TELEGRAPH BNC
BYPASS
OUTPUT. Filtering is applied post-gain. For transmission to external devices, the
m
FILTER TELEGRAPH BNC on the instrument
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.
CAP COMP block contains controls for the adjustment of
The
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
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.
AMPLITUDE control for the FAST component is a ten turn
The
potentiometer with a counting dial and can be used to provide a
FAST pair (AMPLITUDE and TIME CONSTA NT) is
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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, I
OUTPUT, CAP SYNC, EXTERNAL RESET IN, and EXTERNAL COMMAND IN. A 9-pin DIN
connector (for the headstage), a 15 pin I/O
CHASSIS GROUND, and a SPEAKER OUTPUT are also located on the rear panel.
INTERFACE, binding posts for CIRCUIT and
M
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 amplifi er 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 becom e
necessary to independently ground the chassis of the
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.) withi n the bilayer rig,
thus preventing ground loops.
In general, the internal circuitry of the
connected to chassis ground. However, when necessary, the
to tie the circuit and chassis grounds to a common potential. This is the default configuration when the
instrument is shipped from the factory.
BC-535 when it is used as a freestanding devi ce
BC-535 maintains a virtual ground and is not normally not
CIRCUIT GROUND binding post can be used
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BC-535 Preliminary, Rev. 060126 10
Note: We recommend separating the circuit and chassis grounds by di sconnecting the bridging bar
between the associated ground posts. Loosen the posts and slide the bridge to one side.
Gain Telegraph
The GAIN TELEGRAPH i s 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 I
settings are selectable in the front panel
Gain (mV/pA) Gain Telegraph (V)
I
m
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
OUTPUTS block, see page 8)
GAIN are specified below. (Im GAIN
m
Filter Telegraph
The FILT ER 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. (
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
FILTER settings are selectable in the front panel OUTPUTS
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BC-535 Preliminary, Rev. 060126 11
Im output
The Im OUTPUT 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
toggle switch located in the
HOLD control block on the instrument front panel. Use of the rear input
COMMAND 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
square wave and is 100 µs in duration.
SYNC OUT signal is a standard TTL
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
(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.
INPUT and REF
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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
resistor. The precision of this resistor is ± 5%.
MC-1 connects to the two 1 mm mini-jacks on the headstage marked INPUT and REF. The
The
green grounding wire on the
MC-1 contains a 100 pF capacitor connected in parallel with a 1 GΩ
MC-1 is attached to the isolated grounding jack on the headstage.
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Instruments
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 sensi tive electronics in the h eadstage 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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