Warner PC-505B Bedienungsanleitung

PC-505B Manual, Rev. 201013

Warner Instruments

Whole Cell/Patch Clamp Amplifier

Model PC-505B

Warner Instruments

1125 Dixwell Avenue, Hamden, CT 06514

(800) 232-2380 / (800) 547-6766 - support

www.warneronline.com

PC-505B Manual, Rev. 201013

Table of Contents

INTRODUCTION.......................................................................................................................................4

NOMENCLATURE....................................................................................................................................6

Text conventions.....................................................................................................................................6

Device panel abbreviations....................................................................................................................6

Signal polarity conventions....................................................................................................................6

Membrane current (Im).........................................................................................................................6

Membrane potential (Vm) ....................................................................................................................7

CONTROL DESCRIPTION......................................................................................................................8

Front panel..............................................................................................................................................8

COMMANDS block..................................................................................................................................8

FAST CAP COMP (fast capacitance compensation) command block...................................................10

WHOLE CELL COMMAND BLOCK...........................................................................................................10

OUTPUT command block ....................................................................................................................11

METER section ....................................................................................................................................13

Front and rear panel BNC’s and connectors.....................................................................................13

Headstage...........................................................................................................................................16

Electrode holders ...............................................................................................................................16

Model Cell..........................................................................................................................................17

SETUP AND INITIAL TEST..................................................................................................................18

Line voltage...........................................................................................................................................18

Instrument grounding..........................................................................................................................18

Headstage precautions .........................................................................................................................18

Headstage preparation.........................................................................................................................19

Test Procedures ....................................................................................................................................19

Initial connections..............................................................................................................................19

Initial configuration ...........................................................................................................................19

RMS noise checkout............................................................................................................................21

Imoutput checkout ..............................................................................................................................21

Junction zero checkout.......................................................................................................................21

Internal DC voltage command checkout............................................................................................21

External DC Voltage command checkout...........................................................................................22

Internal AC voltage command checkout.............................................................................................23

Auto Zero control checkout................................................................................................................23



Vc commands checkout....................................................................................................................23

PC-505B Manual, Rev. 201013

Cap Comp checkout............................................................................................................................24

Current clamp checkout .....................................................................................................................24

OPERATION............................................................................................................................................26

Initial settings........................................................................................................................................26

Preliminary ...........................................................................................................................................27

Mount the electrode............................................................................................................................27

Submerge the electrode tip.................................................................................................................27

Zero the pipette offset.........................................................................................................................27

Measure the pipette resistance (RP)...................................................................................................28

Voltage clamp........................................................................................................................................29

Attach to the cell and form gigaseal...................................................................................................29

Set electrode capacitance compensation............................................................................................30

Measure seal resistance (RS) .............................................................................................................30

Patch or whole cell recording?..........................................................................................................31

Patch recording - Leak subtraction....................................................................................................31

Patch recording –Excised patch........................................................................................................31

Patch recording - Cell-attached patch...............................................................................................32

Whole-cell recording –Initial conditions...........................................................................................32

Whole-cell recording - Rupture the membrane patch........................................................................32

Whole-cell recording - Series R compensation..................................................................................33

Whole-cell recording - % correction..................................................................................................34

Current clamp.......................................................................................................................................35

Switching into current clamp .............................................................................................................35

Recording in current clamp................................................................................................................35

External command currents ...............................................................................................................36

Bilayer recording..................................................................................................................................36

THEORETICAL CONSIDERATIONS .................................................................................................37

Chloriding silver wire...........................................................................................................................37

Chloriding by electrolysis ..................................................................................................................37

Chloriding chemically........................................................................................................................37

Electrode holders..................................................................................................................................37

Care and use of holders......................................................................................................................38

Cleaning and storage .........................................................................................................................38

Replacing holder parts.......................................................................................................................38

Reference electrodes.............................................................................................................................39

APPENDIX................................................................................................................................................40

Specifications.........................................................................................................................................40

Accessories and replacement parts.....................................................................................................42

Warranty and service...........................................................................................................................43

Warranty.............................................................................................................................................43

Service notes.......................................................................................................................................43

PC-505B Manual, Rev. 201013

Recommended reading.........................................................................................................................44

Glossary.................................................................................................................................................47

PC-505B Manual, Rev. 201013

Warner Instruments

AHarvard Apparatus Company

The PC-505B Whole Cell/Patch Clamp Amplifier is a low noise, resistive-feedback patch clamp

designed for whole-cell, single channel, and bilayer applications. The unique circuitry and

dedicated design of this amplifier allows Warner Instruments to present a superior quality

instrument at a cost significantly below that of many of our competitors.

Principal features of the PC-505B include:

✓Low noise levels of 0.038 pA RMS at 1 kHz

✓Built-in RMS noise monitor

✓Voltage and current clamp modes with independently selectable Vhold and Ihold

✓Bandwidth to 25 kHz

✓Internal 4-pole low-pass Bessel filter with rear panel filter telegraph

✓Built-in test generator

✓Automatic junction potential compensation

✓Two-range fast capacitance compensation

✓Series resistance and C-slow capacitance compensation

✓% Correction circuitry

✓Adjustable duration zap circuit for whole cell membrane penetration

✓Output gain selection with rear-panel gain telegraph

✓Front/rear panel controls and connectors are color coded and organized for quick

reference and convenient operation

THIS EQUIPMENT IS NOT DESIGNED NOR INTENDED

FOR USE ON HUMAN SUBJECTS

PC-505B Manual, Rev. 201013

Warner Instruments

AHarvard Apparatus Company

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 minimize the potential for 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.

➢Warner Instrument product numbers are presented using bold type.

➢References to instrument panel control blocks are specified using UNDERLINED SMALL CAPS.

➢References to specific controls within a block are specified using SMALL CAPS.

➢References to individual control settings are specified using italic type.

➢Special comments and warnings are presented in highlighted text.

Any other formatting should be apparent from context.

Device panel abbreviations

Many controls on the PC-505B have abbreviations associated with them. Several of these

abbreviations are listed here for quick reference. In addition, these and other terms have been

collected and are included in a Glossary at the back of this manual.

Term

Meaning

Sections

CAP COMP

capacitance compensation

FAST CAP COMP

output current

OUTPUT,METER,REAR PANEL

membrane voltage

OUTPUT,METER,REAR PANEL

COMMAND IN voltage

OUTPUT,METER,REAR PANEL

Vc

sum of all command and

compensation voltages

METER

Vc+ h IN

Vcplus HOLDING voltage

METER

P STAT

potentiostat mode

OUTPUT,REAR PANEL

Signal polarity conventions

Membrane current (Im)

Imis indicated as positive when cations flow outward from the pipet tip, through the cell

membrane and into the bath, and/or when anions flow from the bath into the pipet. With

PC-505B Manual, Rev. 201013

Warner Instruments

AHarvard Apparatus Company

outside-out patch and whole cell preparations, this corresponds to the conventional

physiological definition of outward transmembrane current. With inside-out or cell-attached

patches, the physiological transmembrane current equals minus the indicated Im.

Membrane potential (Vm)

Membrane potential is defined as pipet potential minus bath potential. With outside-

out patch and whole cell preparations, this corresponds to the conventional physiological

definition of transmembrane potential. With inside-out or cell-attached patches, the

physiological transmembrane potential equals minus the indicated Vm.

PC-505B Manual, Rev. 201013

Warner Instruments

AHarvard Apparatus Company

CONTROL DESCRIPTION

The instrument front panel is divided into several control blocks. Controls within these

blocks are dedicated towards a common functional purpose. Each control block is outlined in

blue and is clearly labeled as COMMANDS,FAST CAP COMP,WHOLE CELL,and OUTPUT. A final section

contains the LCD METER,as well as the METER SELECT and POWER switches. This section will be

referred to as the METER block.

Front panel

COMMANDS block

The COMMANDS block

contains the VOLTAGE and

CURRENT HOLDING controls, the

JUNCTION ZERO controls, and a

COMMAND SELECT toggle switch

selecting internal or external

command inputs. This block

also contains several controls for

adjusting the sensitivity of input

commands, as well as controls

for the ZAP function.

Voltage and current commands

The VOLTAGE and CURRENT

HOLD controls provide independent modification of holding potential and holding current

settings within the ranges ± 200 mV and ± 1.0 nA, respectively. A MODE toggle switch in the

OUTPUT command block is used to select between voltage clamp (Vc) or current clamp (Ic)

modes. Placing the instrument in current or voltage clamp mode activates either the VOLTAGE

or CURRENT HOLD controls, respectively. This structure allows switching between voltage and

current clamping configurations without the need to readjust settings. VOLTAGE and CURRENT

HOLD controls are not attenuated by COMMAND SENSITIVITY settings.

Internal command, external command, and command sensitivity

The COMMAND SELECT toggle switch selects between an internally generated (internal

command) or an externally generated (external command) command that is applied to the

COMMAND IN BNC located on the instrument rear panel.

NOTE: When set to external command, the COMMAND SENSITIVITY controls attenuate the signal

applied to the COMMAND IN BNC located on the instrument rear panel. When set to internal

command, the COMMAND SENSITIVITY controls select the scaling of the internally generated

TEST PULSE.

The COMMAND SENSITIVITY controls are comprised of an on/off toggle and a sensitivity

selector. The SENSITIVITY SELECTOR attenuates either the internally generated TEST PULSE or

PC-505B Manual, Rev. 201013

Warner Instruments

AHarvard Apparatus Company

any externally applied command voltages connected to the COMMAND IN input BNC by factors

of x0.1, x0.01, and x0.001.

Internal or external commands are activated by moving the COMMAND SENSITIVITY toggle

into the on position. When switched off, these modifiers are disconnected from the COMMAND

pathway.

Test pulse

When internal command is selected and COMMAND SENSITIVITY is switched on, a 1V p-p

square wave test pulse (line frequency; 50/60 Hz) is generated by the PC-505B. The test

pulse is attenuated by the SENSITIVITY SELECTOR toggle and is available for adjustment of

capacitance compensation, measurement of pipet resistance, or for monitoring the formation

of a gigohm seal at the electrode tip.

SENSITIVITY SELECTOR setting

Amplitude of test pulse

square wave (p-p)

x0.1

100 mV

x0.01

10 mV

x0.001

1 mV

When external command is selected, the internal TEST PULSE circuitry is disabled and

commands appearing at the COMMAND IN BNC are attenuated and available to be applied to

the headstage.

Junction Zero and Auto Zero

These controls set the pipette current to zero after the pipette is placed in the test

solution and prior to seal formation. This circuitry is used to compensate for electrode

potentials, liquid junction potentials and other offset voltages, and establishes a zero

baseline reference potential. The 10-turn JUNCTION ZERO control manually adjusts Imbetween



120 mV with immediate response.

With the AUTO ZERO toggle switch in the on position (auto zero LED on) depressing the

associated pushbutton zeros the current automatically. The settling time is rapid when used

with Mpipette resistances, but can take several seconds if used with Gresistances. For

complete compensation, hold the pushbutton in until Imon the METER reads zero.

NOTE: With AUTO ZERO turned on, the manual junction control remains active. In this mode,

depressing the auto zero pushbutton will zero any manual setting, holding potential, and

time-averaged test pulse or command voltage present. Switching AUTO ZERO off restores the

uncompensated current and voltages. To re-zero, repeat using either manual or auto zeroing.

PC-505B Manual, Rev. 201013

Warner Instruments

AHarvard Apparatus Company

Zap

The ZAP function generates an internal +1.5 V pulse which is applied to the headstage.

The pulse duration may be adjusted from 0.1 to 10 ms as indicated. A safe/active toggle

switch arms the circuitry and the pulse is initiated when the ZAP button is depressed.

FAST CAP COMP (fast capacitance compensation)

command block

The two pairs of controls, C-FAST 1/FAST 1and C-FAST

2/FAST 2, are used to adjust the amplitude and time

constant (tau) of the circuitry that compensates for

capacitative currents due to the pipet and other stray

capacitances. This is achieved by applying an appropriate

capacitative countercurrent to the headstage input. These

controls are disabled in current clamp mode.

When properly adjusted, FAST CAP COMP controls have

two important functions: 1) to closely align the voltage

clamp waveform at the cell membrane to the command

signal waveform, and 2) to minimize large current

transients associated with rapid changes in applied

membrane potential. These effects, if uncompensated, can

drive the headstage amplifier into saturation, which can result in the loss of several ms of

data while the headstage recovers.

WHOLE CELL COMMAND BLOCK

The WHOLE CELL command block contains

controls for C-SLOW, SERIES R, %CORRECTION, and

LEAK SUBTRACTION. These controls are used to

compensate for the effects of membrane

capacitance and access resistance when the

amplifier is used in whole cell mode. With the

exception of LEAK SUBTRACTION, these controls are

only available when the PROBE RESISTER (in the

OUTPUT command block) is in low mode.

C-Slow

This control is used to compensate for the

whole–cell membrane capacitance. The

compensated capacitance can be read directly

from the lockable 10-turn control. The associated

toggle switch disables C–SLOW allowing comparison with the uncompensated signal.

This control is not available in patch mode since the FAST CAP COMP circuitry is used to

address capacitances in this mode.

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