Molecular Devices Axoclamp 900A User manual

UserGuidefor
Axoclamp™900A
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Computer‐controlled
MicroelectrodeAmplifier
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MolecularDevicesCorporation
Sunnyvale,California94089
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PartNumber2500‐0179Rev.A
800.998.MATE | www.autom8.com | 650 University Ave # 5, Berkeley, CA 94710 USAt e l 5 1 0 . 8 4 5 . 6 2 8 3 | f a x 5 1 0 . 6 6 5 . 3 9 7 5 | e - m a i l i n f o @ a u t o m 8 . c o m

Copyright
©Copyright2006,MolecularDevicesCorporation.Allrightsreserved.Nopartof
thispublicationmaybereproduced,transmitted,transcribed,storedinaretrieval
system,ortranslatedintoanylanguageorcomputerlanguage,inanyformorby
anymeans,electronic,mechanical,magnetic,optical,chemical,manual,or
otherwise,withoutthepriorwrittenpermissionofMolecularDevicesCorporation,
1311OrleansDrive,Sunnyvale,California,94089,UnitedStatesofAmerica.
Trademarks
Axoclamp900Aisatrademark.DigidataandpCLAMPareregisteredtrademarks
ofMolecularDevicesCorporation.Allothertrademarksarethepropertyoftheir
respectiveowners.
TechnicalAssistance
Fortechnicalassistance,productcommentsandfeedback,orhelptoresolvea
problem,pleasevisit Molecular Devices Technical Support webpage at
www.moleculardevices.com/pages/supportoryoumayreachusbyphoneat
1‐800‐635‐5577(USonly);elsewhere,contactyourlocalrepresentative.
Disclaimer
MolecularDevicesCorporationreservestherighttochangeitsproductsand
servicesatanytimetoincorporatetechnologicaldevelopments.Thisuserguideis
subjecttochangewithoutnotice.
Althoughthisuserguidehasbeenpreparedwitheveryprecautiontoensure
accuracy,MolecularDevicesCorporationassumesnoliabilityforanyerrorsor
omissions,norforanydamagesresultingfromtheapplicationoruseofthis
information.
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iii

Contents
TableofContents
Chapter1:Introduction.................................................................................................................... 1
Chapter2:InstallationandBasicOperation................................................................................ 5
Installation......................................................................................................................................5
CheckList ................................................................................................................................... 5
InstallingHardware .................................................................................................................. 6
InstallingtheAxoclamp900ACommander........................................................................... 6
Input/OutputConnections ...........................................................................................................8
FrontPanel.................................................................................................................................. 8
RearPanel ................................................................................................................................... 9
Overview ......................................................................................................................................11
Axoclamp900ACommanderInterfaceOverview.............................................................. 12
Toolbar ...................................................................................................................................... 12
Meters........................................................................................................................................ 13
ModeButtons ........................................................................................................................... 13
ModeTabs................................................................................................................................. 14
ScaledOutputTabs.................................................................................................................. 15
SettingParametersintheAxoclamp900ACommander.................................................... 15
ToolbarButtonsintheAxoclamp900ACommander......................................................... 18
CommunicationwiththeAxoclamp900A........................................................................... 18
ModelCells...................................................................................................................................19
TheCLAMP‐1UModelCell ................................................................................................... 19
TheMCO‐2UModelCell........................................................................................................ 21
InstallationofModelCells...................................................................................................... 21
v

UserGuideforAxoclamp900A
PowerSupply...............................................................................................................................21
SupplyVoltageSelection........................................................................................................ 21
ChangingtheFuse................................................................................................................... 22
Glitches...................................................................................................................................... 22
GroundingandHum ..................................................................................................................23
Chapter3:SetupandTutorials..................................................................................................... 25
Checklist........................................................................................................................................25
Axoclamp900AandAxoclamp900ACommanderSetup..................................................... 26
Tutorial1:CurrentClamp(IC) ..................................................................................................28
Tutorial2:DiscontinuousCurrentClamp(DCC) ...................................................................34
Tutorial3:DiscontinuousSingle‐ElectrodeVoltageClamp(dSEVC).................................. 37
Tutorial4:Two‐ElectrodeVoltageClamp(TEVC) .................................................................41
I.TEVCUsingtheCLAMP‐1UModelCell.......................................................................... 41
II.TEVCUsingtheMCO‐2UOocyteModelCellandVG‐9ABathClamp ..................... 46
DCRestore................................................................................................................................49
Tutorial5:AutoModeSwitching..............................................................................................50
ExampleofAutoModeSwitching ........................................................................................ 51
SetupofAutoMode ................................................................................................................ 52
Tutorial6:High‐VoltageCurrentClamp(HVIC) ...................................................................55
Chapter4:GuidetoElectrophysiologicalRecording............................................................... 59
GeneralAdvice.............................................................................................................................59
ChamberDesign ...................................................................................................................... 59
Perfusion ................................................................................................................................... 60
MechanicalStability ................................................................................................................ 60
Optics......................................................................................................................................... 61
BathElectrode .......................................................................................................................... 61
InterfacingaComputer........................................................................................................... 62
Noise..............................................................................................................................................62
MeasurementofNoise ............................................................................................................ 62
SourcesofNoise....................................................................................................................... 63
BathErrorPotentials ...................................................................................................................65
(1)MinimizeRb......................................................................................................................... 66
(2)ClampVUsingaBathClamporVirtualGround
b........................................................ 67
(3)IndependentlyMeasureVandSubtractitfromtheCHANNEL1Potential
b........... 68
Summary................................................................................................................................... 70
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Contents
Headstages....................................................................................................................................70
HS‐9ASeries............................................................................................................................. 70
VG‐9ASeries ............................................................................................................................ 70
CurrentClampCircuit ............................................................................................................ 71
MountingtheHeadstage ........................................................................................................ 72
BathConnection....................................................................................................................... 72
StaticPrecautions..................................................................................................................... 72
AcousticPick‐up...................................................................................................................... 72
CompatibilityoftheHS‐2andHS‐2AwiththeAxoclamp900A...................................... 73
GeneralCharacteristics ........................................................................................................... 73
TheMeaningofH.................................................................................................................... 74
GuidelinesforSelectingaHeadstage.................................................................................... 75
HeadstageConnection ............................................................................................................ 77
Interchangeability.................................................................................................................... 77
Cleaning .................................................................................................................................... 77
InputLeakageCurrent............................................................................................................ 77
InputResistance....................................................................................................................... 78
Holders..........................................................................................................................................78
HolderDesign .......................................................................................................................... 78
OptionalAg/AgClPellets ....................................................................................................... 80
HolderUse................................................................................................................................80
HolderMaintenance................................................................................................................ 81
SharpMicroelectrodeRecording............................................................................................... 82
MicroelectrodeProperties....................................................................................................... 82
FillingSolutions ....................................................................................................................... 85
ImpalingCells .......................................................................................................................... 86
TheoryofRecordingModes....................................................................................................... 87
Current‐ClampMode.............................................................................................................. 87
DiscontinuousCurrentClamp(DCC)Mode ....................................................................... 88
DiscontinuousSingle‐ElectrodeVoltageClamp(dSEVC)Mode...................................... 91
NoiseinDCCanddSEVCModes ......................................................................................... 96
TEVCMode .............................................................................................................................. 98
Filters...........................................................................................................................................107
–3dBFrequency..................................................................................................................... 107
TypesofFilters....................................................................................................................... 108
Blanking ......................................................................................................................................110
CurrentMeasurement............................................................................................................... 111
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UserGuideforAxoclamp900A
Virtual‐GroundCurrentMeasurement ..................................................................................112
Chapter5:CommanderReference............................................................................................. 115
AlwaysOnTop ..........................................................................................................................115
BridgeBalance............................................................................................................................ 115
BridgeBalanceOverview ..................................................................................................... 116
BridgeBalanceintheBath.................................................................................................... 118
BridgeBalanceintheCell..................................................................................................... 118
Buzz .............................................................................................................................................119
Clear ............................................................................................................................................120
ConfigureSoftPanel ..................................................................................................................120
DefaultSoftPanelSettings .................................................................................................... 121
HowtoConfigureSoftPanel ................................................................................................ 121
DetectOscillation....................................................................................................................... 123
CurrentClamp ....................................................................................................................... 123
VoltageClamp........................................................................................................................ 124
CurrentInjectionResistor,R0................................................................................................... 124
Holding .......................................................................................................................................124
HowtoUseHolding ............................................................................................................. 125
KeyboardShortcuts................................................................................................................... 125
HotKeys.................................................................................................................................. 125
ShortcutKeys ......................................................................................................................... 125
LoadConfiguration...................................................................................................................128
ModeSwitching.........................................................................................................................129
ConnectinganExternalCommandSignal ......................................................................... 129
AutomaticModeSwitching.................................................................................................. 130
AdditionalNote ..................................................................................................................... 130
MonitorScope ............................................................................................................................131
Options........................................................................................................................................ 131
Options/General....................................................................................................................... 131
ExternalCommandSensitivity ............................................................................................ 132
LowpassFilterType .............................................................................................................. 133
SyncOutput............................................................................................................................ 133
AuxiliaryHeadstageInformation........................................................................................ 134
Miscellaneous......................................................................................................................... 134
Options/Auto............................................................................................................................ 135
SwitchtofromI‐ClamptodSEVC/TEVC......................................................................... 135
viii

Contents
ReturntoCurrentClamp...................................................................................................... 136
Options/Audio .........................................................................................................................137
Mute......................................................................................................................................... 138
Volume.................................................................................................................................... 138
AudioMode............................................................................................................................ 138
AudioSignal........................................................................................................................... 139
Options/QuickSelect............................................................................................................... 139
Options/About .........................................................................................................................140
OutputOffset .............................................................................................................................140
HowtoUseOutputOffset.................................................................................................... 141
AdditionalNotes.................................................................................................................... 142
PipetteCapacitanceNeutralization......................................................................................... 142
InputCapacitance.................................................................................................................. 142
AdjustingCapacitanceNeutralization................................................................................ 143
LimitationsofCapacitanceNeutralization ........................................................................ 144
OscillationDetectionandRemoval..................................................................................... 144
PipetteOffset.............................................................................................................................. 144
HowtoUsePipetteOffset .................................................................................................... 145
AdditionalNotes.................................................................................................................... 145
Pulse ............................................................................................................................................145
HowtoUsePulse................................................................................................................... 146
AdditionalNotes.................................................................................................................... 146
QuickSelect ................................................................................................................................146
ConfigurationFiles................................................................................................................ 146
ExecutableFiles...................................................................................................................... 147
ResettoProgramDefaults........................................................................................................147
ResizeWindow ..........................................................................................................................147
SaveConfiguration.................................................................................................................... 147
ScaledOutput............................................................................................................................. 148
VoltageClamp........................................................................................................................ 148
CurrentClamp ....................................................................................................................... 149
AuxiliaryHeadstages............................................................................................................ 150
Telegraphs............................................................................................................................... 150
ConditioningtheScaledOutput.......................................................................................... 150
HighpassFilter....................................................................................................................... 151
Gain.......................................................................................................................................... 151
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UserGuideforAxoclamp900A
LowpassFilter ........................................................................................................................ 152
SealTest ...................................................................................................................................... 153
HowToUseSealTest............................................................................................................ 153
AdditionalNotes.................................................................................................................... 153
SelectDevice............................................................................................................................... 154
AxoclampTab ........................................................................................................................ 154
SoftPanelTab.......................................................................................................................... 155
SlowCurrentInjection ..............................................................................................................156
HowToUseSlowCurrentInjection ................................................................................... 157
Telegraphs ..................................................................................................................................157
Tuning......................................................................................................................................... 158
HowToUseTuning .............................................................................................................. 158
AdditionalNotes.................................................................................................................... 159
V‐ClampControl ....................................................................................................................... 159
Gain.......................................................................................................................................... 159
Lag ........................................................................................................................................... 159
DCRestore.............................................................................................................................. 160
Chapter6:Troubleshooting ........................................................................................................ 161
VoltageClampProblems.......................................................................................................... 162
dSEVC ..................................................................................................................................... 162
TEVC........................................................................................................................................ 162
AllVoltage‐ClampModes.................................................................................................... 163
SpaceClamp............................................................................................................................... 164
Noise............................................................................................................................................166
Chapter7:Specifications............................................................................................................. 167
FunctionalSpecification............................................................................................................ 167
InstrumentUsage................................................................................................................... 167
MainUnit................................................................................................................................167
Headstage ............................................................................................................................... 168
Axoclamp900AHeadstages................................................................................................. 168
OutputCompliance............................................................................................................... 168
PipetteCapacitanceNeutralization..................................................................................... 168
OscillationDetectionandCorrection.................................................................................. 169
SampleRate ............................................................................................................................ 169
DCCMark/Space ................................................................................................................... 169
PhaseLag ................................................................................................................................169
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Contents
dSEVCGain............................................................................................................................ 169
TEVCGain.............................................................................................................................. 169
DCRestore.............................................................................................................................. 170
Buzz ......................................................................................................................................... 170
Clear(+/–)................................................................................................................................170
StepActivate........................................................................................................................... 170
BlankActivate ........................................................................................................................ 170
Scope........................................................................................................................................ 171
Options.................................................................................................................................... 171
InternallyGeneratedSignals................................................................................................ 172
ExternalCommandInputs ................................................................................................... 173
ExternalCurrentOutputs(2off).......................................................................................... 173
AuxiliaryInputs&Outputs(2off)...................................................................................... 173
ScaledOutputs ....................................................................................................................... 174
Metering(4off)....................................................................................................................... 174
AudioMonitor ....................................................................................................................... 175
PerformanceSpecification........................................................................................................ 175
I‐Clamp(Channels1&2) ..................................................................................................... 175
DCC(Channel1).................................................................................................................... 175
HVIC(Channel2) .................................................................................................................. 176
dSEVC(Channel1)................................................................................................................ 176
TEVC(Channels1&2together).......................................................................................... 176
AppendixA:References.............................................................................................................. 179
AppendixB:CircuitDiagramsRequestForm......................................................................... 183
AppendixC:ImportantSafetyInformation ............................................................................ 185
AppendixD:PolarityConventions ........................................................................................... 193
AppendixE:Glossary................................................................................................................... 195
Index................................................................................................................................................ 197
xi

Introduction
Chapter1:Introduction
Axoclamp900Aisthenewestcutting‐edgeinstrumentfromMolecular
Devices,theworldleaderinhigh‐quality,low‐noisemicroelectrode
amplifiers.LiketheAxoclamp‐2Bthatprecededit,theAxoclamp900A
offersseveralmodesofoperationthatmeasuresignalsfromsinglecells,
tissueslices,andwholeanimalpreparations.Theadvancedsignal
conditioningaddedtotheAxoclamp900Aamplifiersavestheexpenseof
buyingadditionalhardwareandfreesupvaluablespaceinyour
electrophysiologysetup.
BymakingtheAxoclamp900Acomputer‐controlled,itenablesustoadd
severalpowerfulnewfeaturesthatmakeiteasiertosetupandrun
experimentsandthatkeepexperimentsrunninglonger.Thisexcitingnew
instrumentoffersyoutheflexibilityyouneedfortoday’sexperiments,as
wellasforexperimentsyoudecidetodointhefuture.
Thisversatileamplifieroffersseveralmodesofoperation,makingit
suitableforawiderangeofbiologicalpreparationsandformaximum
flexibilityinexperimentaldesign:
 Current‐clamp(I‐Clamp)mode,formeasuringvoltageresponses,is
availableintwochannelswithindependentBridgeBalanceandI=0
options.
1

UserGuideforAxoclamp900A
 Two‐electrodevoltageclamp(TEVC)modeusestwomicroelectrodes,
oneforcontinuousrecordingofelectrodevoltageandtheotherfor
injectionofcurrent.TEVCmodecanbeusedwithlargefrogoocytes
orsmallermammaliancells.Thehighoutputcomplianceofthismode
makesitpossibletovoltageclampcurrentsthatarelargerthan
currentsclampedwithsingle‐electrodetechniques.
 Discontinuouscurrentclamp(DCC)modecanbeusedformore
accuratevoltagemeasurements,andisespeciallyusefulwhensmall
changesinelectroderesistanceoccurduringanexperiment.
 Discontinuoussingle‐electrodevoltageclamp(dSEVC)modeisused
tovoltageclampsmallcellsthatcannottoleratebeingimpaledbytwo
electrodes.Setupcorrectly,thismodeeliminatesthelargeseries
resistanceinherentinmanypreparations.
 High‐voltagecurrentclamp(HVIC)modeisusedprimarilyfor
extracellularisapplications.
Amplifierperformanceisbetterthanever.The±180Voutputcompliance
usedforTEVCandHVICmodesmakesitpossibletopasslargercurrents
andensuresfasterclampspeeds.TEVCanddSEVCmodesbothhavewide
ACvoltage‐clampgainrangesforexcellentvoltagecontrol.WhenDC
Restoreisenabled,theDCvoltage‐clampgainisgreaterthan1,000,000,
ensuringoptimalvoltagecontrolforconstant‐voltagemeasurements.The
newdSEVCdesignismorestableandtwiceasfastasintheAxoclamp‐2B,
providinganexcellentalternativetostandardcontinuoussingle‐electrode
voltageclamping.
TheAxoclamp900Aisessentiallyananaloginput/outputinstrument
similartoconventionalamplifiersbyMolecularDevices.Thus,BNC‐type
inputandoutputconnectionsarenecessarytocommunicatewitha
digitizinginterface,oscilloscopeorotherrecordingdevice.TheAxoclamp
900Acontainsnofrontpanelknobsandswitches.Instead,theinstrumentis
operatedusingacontrolpanelprogram,theAxoclamp900ACommander,
whichrunsonahostcomputerandcommunicateswiththeamplifierviaa
USBcable.
2

Introduction
Computercontrolofthisnextgenerationamplifiermeansthattheknobs,
dialsandbuttonsarenolongerneededandarereplacedbyanintuitive
softwarecontrolinterface.Computercontrolprovidesseveraladvantages
overconventionalamplifiersandmakestheAxoclamp900Aamplifier
easiertouseandenablesexperimentstolastlonger.Automationofseveral
standardtasksbecomespossible,suchasadjustmentofPipetteOffset,
BridgeBalance,andPipetteCapacitanceNeutralization.Oscillationsinthe
measuredsignalcanbedetectedandsuppressedautomatically,inlessthan
1ms,basedonuser‐selectedsettings.Instantswitchingbetweenmodescan
beautomated(I‐ClamptodSEVCorTEVCandbackagain),withusers
definingthethreshold,timing,andtrigger(internalorexternal)formode
switching.InDCCanddSEVCmodes,themonitorsignalusedfortuning
canbedisplayedonthecomputerscreenaswellasonanoscilloscope.
SlowcurrentinjectioncanbeusedinI‐Clampmodetopreventsmall,slow
driftsinthemembranevoltagethatcouldendexperimentsprematurely.
TheAxoclamp900ACommanderinterfaceiscompletelyindependentof
acquisitionsoftware.Thus,theAxoclamp900Acanbeusedwithanydata
acquisitionpackage.ItiscompatiblewithDigidataseriesdigitizers(1200A
orlater)andpCLAMP7(orlater)software.However,telegraphingisonly
supportedinpCLAMPversions10andhigher.Regardingthird‐party
software,seeourwebpage“DeveloperInfo”foradetailedSoftware
DevelopmentKitthatdescribeshowtoreadtelegraphinformation.
Werecognizethatsoftwarecontrolofanamplifierisanunusualstep
forwardforsomeusers.Ifcomputermousecontrolisunsettling,consider
theoptionalSoftPaneldevicetocontroltheAxoclamp900A.TheSoftPanel
isessentiallyahardwareextensionoftheAxoclamp900ACommander
software.Knobsandbuttonsreplacemouseorkeyboardcontrol.Formore
information,visitourwebsiteorcallMolecularDevicesTechnicalSupport.
TheAxoclamp900Aisasophisticatedinstrument.Experiencedand
inexperiencedresearchersalikeareadvisedtoreadthismanualthoroughly
andtofamiliarizethemselveswiththeinstrument.Thefollowingtwo
chaptersprovidestep‐by‐stepinstructionsusingtheCLAMP‐1Uand
MCO‐2Umodelcells.
3

UserGuideforAxoclamp900A
MolecularDevicesispleasedtoansweranyquestionsregardingthetheory
anduseoftheAxoclamp900A.Anycommentsandsuggestionsontheuse
anddesignoftheAxoclamp900Awillbemuchappreciated.Wewelcome
reprintsofpapersdescribingworkperformedwiththeAxoclamp900A.
Keepingabreastofyourresearchhelpsustodesignourinstrumentsfor
maximumusefulness.
4

InstallationandBasicOperation
Chapter2:InstallationandBasicOperation
Installation
Carefullyunpackallparts,andusetheenclosedshippinglisttoverifythat
allpartshavebeenreceived.Retainpackingmaterialsincasethe
instrumentneedstobereturnedtothefactoryatalaterdate.
Fortheinitialcheckout,theAxoclamp900Ashouldbesituatedonabench
topawayfromotherequipment.Donotinstallitinarackuntilthe
checkoutiscomplete.
CheckList
Theseinstallationandcheckoutproceduresrequirethefollowing:
1.Axoclamp900Amainunitwithpowercord.
2.HS‐9AheadstagewithCLAMP‐1Umodelcell.
3.2HL‐Uelectrodeholders.
4.USB(A/B‐type)controlcable.
5.Axoclamp900ACommanderhostsoftware(fromCDorwebsite).
5

UserGuideforAxoclamp900A
6.APCrunningWindows2000orXPProoperatingsystem.ThePC
shouldhaveuptothreespareUSBports:twofortheamplifierand
oneiftheoptionalSoftPanelisused.
7.Externaloscilloscope.
InstallingHardware
1.ConnecttheappropriateendoftheUSBcabletotheCONTROLUSB
connectorontheAxoclamp900Arearpanel,andtheotherendtoa
freeUSBportonyourPC.Thisisthemainconnectionbetweenthe
amplifierandthecomputer.
2.ConnectasecondUSBcablebetweentheSCOPEUSBconnectoron
theAxoclamp900ArearpanelandanotherfreeUSBportonyourPC.
Thisconnectiondeliversthemonitorsignalfromtheamplifiertothe
computerfordiscontinuousmodeoperation.
3.ConnecttheHS‐9Aheadstage(s)totheCHANNEL1HEADSTAGE
andCHANNEL2HEADSTAGErearpanelconnectors.Theamplifier
CANbeleftonwhenchangingheadstages.HOWEVER,DONOT
DISCONNECTHEADSTAGESWHILETHESOFTWAREIS
STARTINGUP.
4.Connectthepowercable,andturnontheAxoclamp900A.Thefront
panelPOWERlightshouldilluminateandthestatuslightshould
eventuallygoout.TheWindowsoperatingsystemautomatically
recognizesthenewUSBhardwareasaHumanInterfaceDevice
(HID).
5.IfyouareusingtheoptionalSoftPaneldevice,connectittoadifferent
computerUSBportusingtheUSBcablesuppliedwiththeSoftPanel.
InstallingtheAxoclamp900ACommander
1.RuntheAxoclamp900ACommanderinstallerfromtheenclosedCD,
orfromtheinstallationfiledownloadedfromtheMolecularDevices
6