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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A Harvard Apparatus Company .

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.
Warner Instruments
A Harvard Apparatus Company .

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.
Warner Instruments
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
Warner Instruments
A Harvard Apparatus Company .

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.
Warner Instruments
A Harvard Apparatus Company .

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
Warner Instruments
A Harvard Apparatus Company .