Warner Instruments BC-535 Bedienungsanleitung

BC-535 Preliminary, Rev. 060126

C-

-5

Warner Instruments

1125 Dixwell Avenue, Hamden, CT 06514

(800) 599-4203 / (203) 776-0664

(203) 776-1278 - fax

BC-535 Preliminary, Rev. 060126

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

Warner Instruments

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

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 .

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