Molecular Devices Axoclamp 900A User Manual Download Page 16 | Manualshive

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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.

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Summary of Contents for Axoclamp 900A

Page 1: ...rode Amplifier Molecular Devices Corporation Sunnyvale California 94089 Part Number 2500 0179 Rev A 800 998 MATE www autom8 com 6 50 Universit y Ave 5 Berkeley CA 9 4710 USA t e l 5 10 8 4 5 6 2 8 3 f...

Page 2: ......

Page 3: ...marks 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 Supp...

Page 4: ......

Page 5: ...iii...

Page 6: ......

Page 7: ...tions 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...

Page 8: ...l 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 Vol...

Page 9: ...eability 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 Micro...

Page 10: ...illation 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...

Page 11: ...ation 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 Addit...

Page 12: ...l 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 Chapte...

Page 13: ...73 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...

Page 14: ......

Page 15: ...ble 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...

Page 16: ...ular 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 a...

Page 17: ...e 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...

Page 18: ...ry 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 Axocla...

Page 19: ...he 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...

Page 20: ...er 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...

Page 21: ...days to check for a new download 3 Next you see the Select Device dialog In the Axoclamp tab select Axoclamp Hardware then click the Scan button The amplifier serial number is identified in the list...

Page 22: ...is enabled when the user has checked one of the Switch to dSEVC TEVC on ext input checkboxes in the Headstage 1 or 2 section at the top of the Axoclamp 900A Commander For example a TTL Low 0 0 8 V in...

Page 23: ...rent IC External Command Potential VC or External Command Current IC Auxiliary Potential if HS 9A headstage attached or Auxiliary Current if VG 9A headstage attached The same inputs and outputs are av...

Page 24: ...e connectors with an appropriate optional adapter cable 1 2100 0934 10AUX1 OUTPUT and 10AUX2 OUTPUT These BNC outputs provide 10 output signal for the respective AUXILIARY HEADSTAGE channels REMOTE Mo...

Page 25: ...puter to control the Axoclamp 900A SCOPE USB Connects to a USB 2 0 port of the host computer to provide Scope window data This is a self contained single channel 5 MHz acquisition system that allows t...

Page 26: ...amp 900A Commander Interface Overview The Axoclamp 900A Commander front panel is designed to resemble hardware instrument controls It is divided into five main sections Toolbar Meters Mode buttons Mod...

Page 27: ...hannel This can be activated in either voltage clamp or current clamp modes but not I 0 mode or with slow current injection Note that to measure resistance the Axoclamp 900A Commander generates a comm...

Page 28: ...utput setting on the Options General tab External Mode Control The mode buttons can be operated from an external source Check the Switch to dSEVC on ext input or Switch to TEVC on ext input checkbox t...

Page 29: ...s in the Axoclamp 900A Commander User configurable numerical fields on the Axoclamp 900A Commander printed in blue text are called gliders Placing the mouse over a glider changes the cursor to Click t...

Page 30: ...hift boosts sensitivity by 10 and Ctrl by 100 then at Medium setting they increase sensitivity 20 and 200 times respectively while with a basic Coarse setting these are 50 and 500 times respectively K...

Page 31: ...ending on the value displayed e g the Tuning amplitude field displays some values in pA and others in nA When typing in values you must always express the value in terms of the smaller unit You can ca...

Page 32: ...y to toggle between window sizes Most other buttons are self explanatory with the possible exception of the Save Configuration F5 Load Configuration F4 and Quick Select buttons These buttons allow the...

Page 33: ...1 I Clamp LED and the blue TEVC LED should light Check to see that the LEDs change with the modes selected in the software 4 If the USB cable is unplugged the STATUS LED flashes briefly ON then OFF M...

Page 34: ...hniques and offset adjustment When the switch is in the CELL position both microelectrode resistors are effectively intracellular In Bridge or DCC mode you should see exponential voltage responses to...

Page 35: ...ctrodes The circuit diagram is given below Figure 4 MCO 2U model cell Installation of Model Cells To install the model cell plug it into one or both of your headstages Connect the gold plated ground j...

Page 36: ...pply transients glitches Although normally inconsequential glitches could cause transients to appear on the voltage and current outputs that may corrupt high sensitivity recordings The most effective...

Page 37: ...to radiate into its electronics A thick sheet of steel placed between the Axoclamp 900A and the radiating equipment can effectively reduce the induced hum 3 Initially make only one connection to the A...

Page 38: ...6 Experiment While hum can be explained in theory e g direct pickup earth loops in practice the ultimate theory is the end result Following the rules above is the best start The final hum level can of...

Page 39: ...orial procedures require the following Axoclamp 900A main unit with power cord Two USB control cables Axoclamp 900A Commander software A PC running Windows 2000 or XP Professional External two channel...

Page 40: ...Axoclamp 900A the associated computer and an oscilloscope in a standalone fashion on a bench top The tutorials may also be performed at a later date with the equipment mounted in a typical laboratory...

Page 41: ...ge The switch should be in the BATH position 9 Place the headstage and model cell into a shielded enclosure optional ensuring that the shield is also connected to the signal ground 10 Turn the Axoclam...

Page 42: ...Connect another BNC cable between the SCALED OUTPUT for Channel 2 and the oscilloscope 16 Connect another BNC cable between the external trigger input of the oscilloscope and the SYNC OUTPUT BNC on t...

Page 43: ...wer ON 7 Run the Axoclamp 900A Commander software 8 Press the Reset to Program Defaults button in the toolbar at the top of the Commander and accept the warning Information about the connected headsta...

Page 44: ...M If not click on the Lock button and press the Auto button again You should also be able to click on the Bridge resistance field and use the software glider to adjust the bridge balance resistance No...

Page 45: ...ated electrode capacitance Figure 9 Bridge Balance of voltage signal in bath 14 Check the Pipette Capacitance Neutralization checkbox Change the value in the numeric field until the capacitive transie...

Page 46: ...should not be done if this tutorial is being performed solely in preparation for another tutorial Oscillation Detection 16 Click Detect Oscillation and select Disable Neutralization Figure 12 Oscillat...

Page 47: ...on checkbox Note Oscillation Detection is available in current clamp modes of IC and DCC but not HVIC It is also available in the two voltage clamp modes TEVC and dSEVC Slow Current Injection 17 Slow...

Page 48: ...nstant is shorter than the test waveform frequency Change the slow current Time constant to 2 seconds Deselect and reselect Inject slow current Notice that the speed of establishing the average DC lev...

Page 49: ...compared with I Clamp mode with Bridge Balance is that it is tolerant of small changes in microelectrode resistance The disadvantage is that DCC mode is noisier than I Clamp mode During DCC mode CHANN...

Page 50: ...shape to the IC waveform except it will have about double the noise Figure 18 Membrane potential signal in DCC mode 4 Adjust the Pipette Capacitance Neutralization value and Rate in the I Clamp 1 tab...

Page 51: ...compensated Figure 20 Pipette Capacitance Neutralization over compensated in DCC mode Tutorial 3 Discontinuous Single Electrode Voltage Clamp dSEVC dSEVC mode is used to voltage clamp small cells that...

Page 52: ...ted the monitor waveform is displayed in the Scope window on the computer screen Figure 21 Monitor signal for dSEVC mode The important feature is that the voltage transients decay completely by the ti...

Page 53: ...e with about a 20 ms rise time Note that we programmed a voltage of 10 mV but only got a 6 mV response indicating a higher gain is required Also the current trace IN1 shows a similar settling time In...

Page 54: ...V and the rise time should be less than 300 s with noise around 10 mVp p See the expanded time scale below Figure 24 Voltage and current signals for dSEVC mode with further tuning 8 With practice you...

Page 55: ...tion Figure 26 Monitoring the dSEVC voltage response with a second headastage IN0 is the membrane voltage as measured by Headstage 1 dSEVC mode IN1 is the membrane voltage as measured by Headstage 2 w...

Page 56: ...UT to oscilloscope channel 2 3 Press the Reset to Program Defaults button and accept the warning 4 Connect a Headstage 1 to the ME1 input of the CLAMP 1U model cell b Headstage 2 to the ME2 input of t...

Page 57: ...10 mV mV b Channel 2 Headstage 2 0 1 Membrane Current 1 V A 10 Adjust the Gain until channel 1 starts to overshoot The Rise time should be about 200 s Figure 27 Voltage and current signals for TEVC m...

Page 58: ...e membrane current 1 V A The voltage clamp Gain is 388 Pipette Capacitance Neutralization is 3 5 pF Oscillation Detection 1 In the Detect oscillation section of the TEVC tab you can choose to Reduce g...

Page 59: ...f a typical oscillation showing that it was detected and removed in about 2 ms Figure 31 Automated suppression of voltage signal oscillation in TEVC mode IN0 is 10 the membrane voltage Note Oscillatio...

Page 60: ...gand gated channels low membrane resistance can lead to loss of tight voltage clamp when high current is passed This procedure explains how to overcome each of these problems Equipment required MCO 2U...

Page 61: ...ton and accept the warning 6 Verify that the R0 value for Headstage 1 is 10 M and the R0 value for Headstage 2 is 1 M 7 Zero the Pipette Offset for Channels 1 and 2 outlined in the Current Clamp secti...

Page 62: ...set the Lag to about 0 2 ms 14 Select TEVC mode 15 For the SCALED OUTPUT signals select a Channel 1 Headstage 1 Membrane Potential 10 mV mV b Channel 2 Headstage 2 0 1xMembrane Current 0 1 V A 16 Turn...

Page 63: ...ring the voltage pulse increases channel 2 on the oscilloscope by about 450 mV 4 5 A and the voltage clamp voltage drops to about 48 mV due to the extra load DC Restore DC Restore provides optimal vol...

Page 64: ...ddition to manually controlled mode switching from Axoclamp 900A Commander Axoclamp 900A has the ability to automatically switch between I Clamp and V Clamp modes and back again Auto mode switching al...

Page 65: ...lowing switches from I Clamp to TEVC mode when membrane voltage in headstage 1 passes through 20 mV in a negative to positive direction It stays in TEVC mode for 500 ms generating a 0 mV to 10 mVp p 1...

Page 66: ...mp 2 tab set the TUNE input for Headstage 2 to 1 nA 6 Oscilloscope Channel 1 should show a 50 400 mV triangular wave with rounded sides This equates to a 5 40 mV waveform that will trigger the mode ch...

Page 67: ...rn to I Clamp after 500 ms In order to monitor mode changes on the scope we need to program the SYNC BNC to follow mode changes 7 Click on the General tab 8 In the Sync Output group click the Mode rad...

Page 68: ...veforms should result Figure 36 Automated Mode Switching signals IN0 is 10 the membrane potential in Channel 1 IN1 is the Mode signal HIGH is V Clamp mode and LOW is I Clamp mode Explanation of the wa...

Page 69: ...o the preparation or electrode resistance is very high For example if you want to pass 1 A current into the CLAMP 1U model cell it requires more than 100 V of dynamic range for the driving electrode T...

Page 70: ...al selected should be Headstage 2 0 1 Membrane Potential 9 Connect scope channel 2 to SCALED OUTPUT 2 10 The Channel 2 SCALED OUTPUT Signal selected should be Headstage 2 0 1 Membrane Current Figure 3...

Page 71: ...lect Membrane Potential 1 mV mV Figure 38 Bridge Balance settings 14 Check Bridge Balance and balance bridge 50 M Figure 39 HVIC mode with Bridge Balance 15 HVIC with Bridge Balance 50 1 M The 1 ms sp...

Page 72: ......

Page 73: ...perienced researchers working in laboratories around the world However as is the case for all advice and particularly that pertaining to research the suggestions given here should be taken as provisio...

Page 74: ...s and neither are they tolerant of large drifts in the tissue or electrode that tend to pull the electrode out of the cell For this reason it is important to use a good drift free micromanipulator for...

Page 75: ...00A Commander This displays on the meter the pipette voltage required for zero current through the electrode If for instance the meter shows 2 mV there has been a 2 mV drift since the electrodes were...

Page 76: ...rd that is provided with the interface Finally it is necessary to install software to control the interface Software is available from Molecular Devices pCLAMP or other vendors or you can write your o...

Page 77: ...resistive load the noise is given approximately by 12 R Vrms 10 kHz bandwidth where R is the parallel combination of the current injection resistor R0 and the load resistance i e the electrode resista...

Page 78: ...determined by the effectiveness of the Capacitance Neutralization circuit Compensation Circuits V Clamp Increasing Voltage Clamp Control Gain is required for optimal voltage control and bandwidth but...

Page 79: ...ng very large cells such as Xenopus oocytes in which case the ionic current may be of the order of several microamperes or even several tens of microamperes Depending upon the method of grounding the...

Page 80: ...it is relatively small On the other hand the resistance of the grounding pellet and agar bridge are larger but one can take precautions to minimize them A 1 mm diameter Ag AgCl pellet in Ringer s sol...

Page 81: ...can actually measure Rb in Bridge mode by placing both micropipettes in the bath Put the micropipette connected to CHANNEL 1 on the far side of the micropipette connected to CHANNEL 2 away from the ba...

Page 82: ...espective of the voltage drop across Rb1 This configuration is particularly recommended for voltage clamping oocytes The VG 9A 100 bath clamp is required for the fastest responses and guaranteed stabi...

Page 83: ...and subtracted from the measurements made by CHANNEL 1 and CHANNEL 2 The voltage recorded by the bath microelectrode is available at the SCALED OUTPUT signal BNC on the front panel You can select thes...

Page 84: ...th each Axoclamp 900A amplifier HS 9A 0 1 and HS 9A 1 Other headstages may be substituted on request at the time of purchase All headstages record voltage at unity gain Several headstage current gains...

Page 85: ...f which is the pipette voltage Vp A2 is a summing amplifier used for injecting current into the cell The voltage across the headstage resistor R0 is equal to Vcmd regardless of Vp Thus the current thr...

Page 86: ...old plated 1 mm plug on the rear of the headstage The bath should not contact any other ground e g Signal Ground Static Precautions The headstage can normally be safely handled However if you are in a...

Page 87: ...one the Axoclamp 900A Commander recognizes the headstage as a custom type and prompts for current injection resistance and capacitance values This message appears on startup of the Commander Once a cu...

Page 88: ...nsitivity to current commands the sensitivity of the current monitors and the gain in SEVC mode Since the effects always appear in multiples of ten they are easy to calculate For your convenience Tabl...

Page 89: ...es use the largest feasible H value This is because the current passing response is best with low values of R0 2 A limitation on using large H values is that as R0 becomes smaller the input leakage cu...

Page 90: ...uate performance even if R0 is as small as 1 10 or as large as 10 Re USE RECOMMENDATION NORMAL INTRACELLULAR BRIDGE RECORDING Follow the general rule EXTRACELLULAR RECORDING Since there is no current...

Page 91: ...Avoid spilling liquids on the headstage Input Leakage Current All DC connected systems suffer from the problem of drift With changes in temperature and the passage of time the DC transfer functions of...

Page 92: ...er possible factors which would decrease the input resistance are minimized For example the field effect transistor FET input of the headstage is referenced to the input voltage rather than to ground...

Page 93: ...r The holder mates with the threaded Teflon connector on U type Molecular Devices headstages and is secured in place with a threaded collar Figure 44 Exploded view of the HL U holder The bore size of...

Page 94: ...inner diameter ID of the pipette must be 1 mm A wax sealed Teflon tube surrounds the silver wire This ensures that the electrode solution only contacts the Ag AgCl pellet Three pellet assemblies are s...

Page 95: ...er wire down to the bare metal using fine sand paper and immerse the cleaned wire in bleach for about 20 minutes until the wire is uniformly blackened This provides a sufficient coat of AgCl to work r...

Page 96: ...ffer advantages In this chapter it is assumed for the most part that sharp microelectrodes are being used for the I Clamp recording Microelectrode Properties Users of sharp microelectrodes spend far m...

Page 97: ...tance The settling time of a microelectrode depends not only on Re but also on the transmural capacitance Ct from the inside of the microelectrode to the external solution For fastest settling Ct must...

Page 98: ...terial This can be done just before use by dipping the filled microelectrode into a fluid such as silicone oil or mineral oil Another method is to coat the microelectrode with Sylgard 184 or Q dope mo...

Page 99: ...croelectrode is pressed into an unvented holder The suction port on the HL U series holders provided with the Axoclamp 900A should therefore be left open Using filling solutions with low pH or adding...

Page 100: ...ton to null the offset Check the stability of the bath electrode and microelectrode Drifts in Scaled Output Membrane Potential indicates that the electrode wires probably need to be rechlorided Also c...

Page 101: ...arger reflecting the membrane time constant and input resistance After impaling the cell it is often helpful to back off the microelectrode slightly and allow the penetration to stabilize for a few mi...

Page 102: ...HANNEL 1 is memorized by a sample and hold circuit in between each current passing period after all transient voltages due to current passing have decayed Thus the membrane potential can be recorded i...

Page 103: ...Vcmd position This connects Vcmd to a differential amplifier A4 arranged so that its output is V1 Vcmd The voltage appearing across R0 is exactly equal to Vcmd thereby forcing the current I0 into the...

Page 104: ...e of this current V1 rises The rate of rise of V1 is limited by the parasitic effects of capacitance through the wall of the glass microelectrode to the solution and capacitance at the input of the bu...

Page 105: ...and this average value is available on the Scaled Output Headstage 1 Membrane Current signal During DCC mode the input to the CCS and the output of the CHANNEL 1 current monitor are automatically sca...

Page 106: ...ifier A2 The output of this amplifier becomes the input of a controlled current source CCS if the switch S1 is in the current passing position The gain of the CCS is GT The CCS injects a current into...

Page 107: ...ach cycle and voltage recording for 70 of each cycle The cycling rate sample rate must be chosen so that there are ten or more cycles per membrane time constant This enables the membrane capacitance t...

Page 108: ...is too slow the dSEVC becomes unstable This is because the long current passing period charges the membrane potential past the desired potential before the clamp has an opportunity to take a new sampl...

Page 109: ...f G will have to be used to maintain stability Clamp Error With finite gains in the voltage clamp circuit Vm does not quite follow Vc The error is Vcmd Vm Similarly if Vcmd is constant and the cell me...

Page 110: ...orded The added consequence is that Rs limits the maximum rate at which the membrane can be charged To eliminate Rs altogether watch the MONITOR output and make sure the transient decays completely be...

Page 111: ...where primary efforts have been taken to keep the input capacitance low to much larger factors in systems where large amounts of capacitance to earth and capacitance to shield are tolerated The secon...

Page 112: ...he records is by averaging repetitive responses This well known procedure reduces the noise by the square root of the number of averages without affecting the time course of the signal Although the no...

Page 113: ...e Pipette Capacitance Neutralization setting of CHANNEL 1 affects the voltage clamp response The Seal Test setting delivers a voltage pulse The best settings of the voltage clamp parameters are found...

Page 114: ...ted using microelectrodes of comparable resistance even given the higher frequency response of the TEVC The major drawbacks to TEVC systems are that two microelectrodes must be applied to one cell and...

Page 115: ...a detailed discussion of this type of series resistance and the methods used to minimize it see Bath Error Potentials on page 65 Selection of the Membrane Voltage Recording Headstage CHANNEL 1 Usually...

Page 116: ...ugh compliance to fully resolve these large transients Table 4 summarizes the current passing capability of the HS 9A headstages Headstage R0 M Short Circuit Current Conversion HS 9A 0 1 100 1 8 A 100...

Page 117: ...the current passing microelectrode Tuning the Voltage Clamp Fidelity and Stability The object of tuning the voltage clamp is to maximize the feedback gain There are two reasons first the bandwidth of...

Page 118: ...xponentially towards its new value For K 1 the time constant for the relaxation is C R m e 2 Increasing the clamp gain decreases the time constant for the step response For example if Re2 10 M Cm 1000...

Page 119: ...1 Inter Electrode Coupling Capacitance and Shielding When the voltage recording microelectrode and current passing microelectrode are applied to a cell there is considerable coupling capacitance Cx be...

Page 120: ...the high frequency measurement of the current unless the capacitance neutralization of CHANNEL 2 is properly set Under these circumstances the capacitance neutralization of CHANNEL 2 may have a signif...

Page 121: ...chnique such as measuring the current using the macropatch technique might be considered It is good practice to keep the tips of the two intracellular microelectrodes as far apart as possible inside t...

Page 122: ...the Scaled Output of the Axoclamp 900A are 4 pole filters Filters with more poles can be constructed but they are more complex to implement and yield diminishing returns Figure 48 Filter characterist...

Page 123: ...ortant considerations when selecting the filter cutoff frequency Aliasing If the digitizing interface samples at 2 kHz for example any noise in the sampled signal that has a frequency greater than 1 k...

Page 124: ...han 0 4 ms so the action potentials are minimally distorted by the filter According to the above equation then the appropriate filter setting would be 10 kHz In practice you may need to make other com...

Page 125: ...ing period ends Usually this deviation is preferable to the situation that can occur if Blanking is not used If Blanking is not used the artifact picked up by CHANNEL 1 is treated by the voltage clamp...

Page 126: ...le current into the bath can be separately measured using a virtual ground headstage see below Virtual Ground Current Measurement A Virtual Ground headstage can be used to ground the preparation bath...

Page 127: ...ent as line frequency noise in the current signal Saline filled tubing acts as an excellent antenna To prevent them carrying hum long saline filled tubes should have the saline pathway broken by an ai...

Page 128: ......

Page 129: ...dix describing Polarity Conventions This summarizes the conventions used for the polarities of currents and voltages in all amplifiers manufactured by Molecular Devices Always On Top Always On Top F3...

Page 130: ...s the adjustable field to the resistance it is compensating for and checks the checkbox Auto can be used whether or not Bridge Balance has already been activated You can fine tune the resistance value...

Page 131: ...epped to a new value top there is a rapid voltage step on Vp due to the ohmic voltage drop across the microelectrode middle Following this instantaneous step there is a slower rise in Vp largely due t...

Page 132: ...nuously through the experiment by injecting a small current step near the beginning of each data sweep It is recommended that Pipette Capacitance Neutralization be set at the same time as Bridge Balan...

Page 133: ...on is achieved by increasing the Pipette Capacitance Neutralization by a small amount over the currently set level Therefore it is essential that you set Pipette Capacitance Neutralization to the corr...

Page 134: ...one extracellularly Penetrating cells Sometimes microelectrode tips press against the cell membrane but fail to penetrate A quick press on the Clear buttons will often force the electrode to penetrate...

Page 135: ...the device the SoftPanel tool button is disabled Default SoftPanel Settings See below for the default SoftPanel settings for the Axoclamp 900A These settings can be customized to the individual user F...

Page 136: ...meter that is currently selected in Commander in increments equivalent to a knob click with Shift Page Down Decreases the parameter that is currently selected in Commander in increments equivalent to...

Page 137: ...s you with options to protect your cell from harmful oscillations during a current clamp experiment by automatically disabling or reducing Pipette Capacitance Neutralization Use the Detect Oscillation...

Page 138: ...are suppressed by changing the Gain setting Current Injection Resistor R0 The current injection resistor in the headstage is automatically detected and displayed in the R0 field at the top of the Hea...

Page 139: ...essed in units of nA Check the Holding checkbox to apply the holding current or potential Settings can be adjusted after the command has been activated Uncheck the checkbox to stop the holding current...

Page 140: ...ope F11 Channel 1 Scaled Output Scope Shift F11 Quick Select Button 1 Ctrl 1 Quick Select Button 2 Ctrl 2 Quick Select Button 3 Ctrl 3 Meters and Mode Selection Headstage 1 Total Resistance Rt Ctrl R...

Page 141: ...ro Scaled Zero Ctrl Shift O letter V Clamp Tab Zero Scaled Output Ctrl Shift O letter Pulse Ctrl Shift P Keystroke Function Adjustable Numerical Fields Up Arrow Increases value For dual control field...

Page 142: ...active field Ctrl Tab Go to next tab Shift Ctrl Tab Go to previous tab Arrow Move between options in non numerical fields Adjust values in numerical fields see above Adjustable Numerical Fields Space...

Page 143: ...when it is configured for external switching allows you to switch between current and voltage clamp modes with a remote device e g a Clampex digital output for one channel at a time Alternatively the...

Page 144: ...mbrane potential threshold is configured on the Options Auto tab When automatic mode switching is configured the Mode Switch label changes to Switch to dSEVC TEVC at threshold but the automatic switch...

Page 145: ...ignal when the Shift key is held down The corresponding shortcut is Shift F11 Options The Options F10 dialog box has five tabs on which many Commander parameters are set The Options tabs are Options G...

Page 146: ...gs 20 mV V and OFF 20 mV V means that a 1 V step applied to the COMMAND BNC appears to the cell as a 20 mV step i e external commands are divided down by 50 fold This setting should be used when you w...

Page 147: ...utput Lowpass Filter Sync Output The SYNC OUTPUT BNC on the rear panel of the Axoclamp 900A outputs a TTL signal HIGH 2 5 V LOW 0 0 8 V synchronized with the feature selected in this section This can...

Page 148: ...tion reports the headstage types and the gain they apply prior to gain applied with the gain settings in the Axoclamp 900A Commander interface Signals from both auxiliary headstages are available for...

Page 149: ...return to current clamp after a change to voltage clamp are at the bottom of the section Changes are not triggered from I 0 mode You can synchronize a TTL output signal to amplifier mode selection wi...

Page 150: ...Switch to dSEVC TEVC at threshold You must configure the delay field below for any of these selections and configure the membrane potential threshold field if you have selected a threshold option Dela...

Page 151: ...tion However you must ensure that when the Axoclamp first switches to voltage clamp the external TTL signal is HIGH so that a change to LOW triggers a return to current clamp at the correct time Manua...

Page 152: ...he Axoclamp 900A with the possibility of causing damage to its circuitry Mute Check this option to mute the audio monitor Volume Use the slide to set the volume of the audio monitor right to increase...

Page 153: ...output for each channel in each mode Whether you hear Channel 1 or Channel 2 at any time depends on which tab is uppermost on the front panel If a Channel 1 tab is on top the Channel 1 output is audi...

Page 154: ...nd DSP Firmware This information is automatically displayed A link to the Axoclamp 900A Commander Update page at Molecular Devices web site Users are advised to check this page at regular intervals to...

Page 155: ...The Zero Output Offset only affects the signal on the Scaled Output In other words the cell is not affected by the Output Offset command No other input or outputs are affected The offset is reported...

Page 156: ...ound of the microelectrode and any connecting lead Cin2 Cin combined with the microelectrode resistance Re acts as a lowpass filter for signals recorded at the tip of the microelectrode For optimal pe...

Page 157: ...ssible The technique is known as bootstrapping Unity gain feedback is used to reduce the component of stray capacitance that exists between the amplifier input and its power supplies and case Sophisti...

Page 158: ...ce the neutralizing circuit adds noise to the voltage signal It is important to recognize that the capacitance neutralization circuit is not more than 90 effective even for ideal microelectrodes This...

Page 159: ...250 mV The glider control allows fine adjustments with increments of 20 V for current clamp To reset the Pipette Offset click on the locked padlock button to unlock it and click the Zero button again...

Page 160: ...onal Notes Pulse generates a Sync Output Quick Select The three Quick Select buttons can be customized to Open saved Configuration files acc of Axoclamp 900A Commander Run an executable file on your c...

Page 161: ...2 resizes the Axoclamp 900A Commander interface It toggles the interface between three states Full display the whole interface is shown Meter panel only the toolbar meters and mode buttons are shown M...

Page 162: ...with Load Configuration Quick Select or by using a desktop shortcut command line switch See Load Configuration Scaled Output The Scaled Output field is located in the lower part of each of the I Clam...

Page 163: ...note below Auxiliary 2 Current Current from the rear panel AUXILIARY HEADSTAGE 2 connection see note below Current Clamp The signals available in current clamp are Membrane Potential Membrane Potenti...

Page 164: ...xiliary headstages at the AUXILIARY 1 HEADSTAGE and AUXILIARY 2 HEADSTAGE inputs typically as headstages for extracellular recordings The scale factor determined by the headstage is displayed beside t...

Page 165: ...m the right click popup menu to set the cutoff frequency desired Filtering begins immediately Set the field to DC to bypass this filter Gain Scaled Output Gain is in the Scaled Output section in the l...

Page 166: ...e filter type The Axoclamp 900A Commander offers a choice of two different lowpass filters to the Scaled Output Bessel or Butterworth Select these on the Options General tab Different cutoff frequency...

Page 167: ...e in order to monitor some parameter in your experimental setup You can choose potentials from 0 to 1000 mV Discrete frequency settings between 2 and 5000 Hz are available View the options by right cl...

Page 168: ...900A Commander software to your Axoclamp 900A amplifier If you want to run more than one Axoclamp 900A amplifier at a time simply open a new instance of the Axoclamp Commander for each amplifier and s...

Page 169: ...l Tab Use this tab to associate a SoftPanel with Axoclamp 900A Commander Scan Click the Scan button to search all serial and USB ports for SoftPanels Results are displayed in the Port field Once you h...

Page 170: ...ng The speed at which current is injected into the cell depends on the current injection resistor and load as well as the setting in the Time constant field The time constant values shown in the glide...

Page 171: ...ange of its parameter settings These telegraphs are handled by the program using the Windows messaging system The benefit of this is that no additional telegraph cabling is required the computer conne...

Page 172: ...current injection resistor selected Injection Resistor Current Amplitude 1 M 12 A 10 M 1 2 A 100 M 120 nA Table 8 Headstage current amplitude range Frequency settings for Tuning range in discrete valu...

Page 173: ...cy response of the voltage clamp circuit can be modified by the Lag control The voltage clamp circuit can thereby be adjusted to compensate for the non ideal phase response of real membranes The contr...

Page 174: ...but variable input leakage current is due to DC current flow through the dielectric of the capacitor C used for pipette capacitance neutralization For example the pipette potential might be 200 mV al...

Page 175: ...nected to the Axoclamp 900A except for the oscilloscope Ideally remove the Axoclamp 900A from the rack Work through the Seup and Tutorials This can often uncover a problem that is in your setup If the...

Page 176: ...se The inter electrode coupling capacitance may be too high Suggestion Check that the shield between the microelectrodes is correctly placed and adequately grounded Symptom The voltage clamp is slow t...

Page 177: ...quired to drive the same current If the problem lies in the virtual ground unit use another one with higher range All Voltage Clamp Modes Symptom Unable to offset the voltage microelectrode voltage to...

Page 178: ...causes 1 A large series resistance or 2 the cell is not isopotential The latter is common when you voltage clamp a neuronal soma with an axon and dendrites attached The slower components in the curre...

Page 179: ...istorted As a general rule the voltage clamp is considered to be acceptable if the length of the attached axon or dendrites is no more than 0 1 length constants Even this short length will cause signi...

Page 180: ...contributing noise sources must be minimized Specifically the headstage the pipette glass the holder and in the case of patch pipettes the membrane seal contribute significant noise even under circum...

Page 181: ...ss otherwise specified TA 20o C 1 hr warm up time Main Unit Line voltage universal 100 240 VAC Line frequency 50 60 Hz Power 30 W maximum Fuse 5 mm 20 mm 250 V 2A slow Case H W L 4 3 19 14 3 109 mm 48...

Page 182: ...0 10 M HS 9A 10 unity gain voltage follower R0 1 M VG 9A 10 bath clamp R0 1 M VG 9A 100 bath clamp R0 0 1 M Ro is the current passing resistor in the headstage Output Compliance CHANNEL 1 12 V in all...

Page 183: ...n I Clamp and DCC Oscillation correction reduces neutralization by 1 pF Optionally it can reduce neutralization to zero SEVC and TEVC Oscillation correction reduces voltage clamp gain by 2 1 Optionall...

Page 184: ...manual up to 0 5 s Step Activate Available on each channel STEP ACTIVATE 1 and STEP ACTIVATE 2 inputs work independently setting external timing for going between the holding level and a programmable...

Page 185: ...ate 2 kHz to 5 MHz Resolution 14 bits Monitor signal Raw membrane potential before sample and hold PC display PC digital scope that is launched whenever DCC or dSEVC is selected It can be inhibited an...

Page 186: ...ogrammable 20 ms to 2 s Test level Up to 10 R0 e g 10 A for HS 9A 10 Test Frequency 2 Hz to 5 kHz includes exact 50 Hz and 60 Hz Holding level Up to 1 R0 e g 1 A for HS 9A 10 dSEVC Test level Up to 4...

Page 187: ...el 1 I CLAMP Command BNC Command gain V 10 R0 e g 100 nA V for HS 9A 10 TEVC V CLAMP Command BNC Command gain 20 mV V HVIC Channel 2 I CLAMP Command BNC Command gain V R0 e g 1000 nA V for HS 9A 10 Ex...

Page 188: ...or VG 9A 100 Output Calibration 1 A V for VG 9A 100 Since there is a fixed gain of 10 1 the BNC output saturates at 1 10th of the maximum current but clamp continues to operate up to the maximum speci...

Page 189: ...d Oscillator VCO VCO 100 mV 500 Hz 0 mV 2400 Hz 100 mV 4000Hz Level Programmable volume up to 1 Vp p Output Impedance 500 short circuit protected Noise 1 mVrms Performance Specification I Clamp Channe...

Page 190: ...ent settling time2 500 s to 10 of peak value Voltage noise2 180 Vrms Current noise2 0 30 nArms TEVC Channels 1 2 together Internal holding level 200 mV AC voltage clamp gain 20 to 50 000 Voltage clamp...

Page 191: ...rent noise 70 nArms 3 Model cell with two 1 M resistors to simulate the electrode resistances and a 1 M resistor and 220 nF capacitor in parallel to simulate the oocyte membrane Lowpass filter 10 kHz...

Page 192: ......

Page 193: ...1988 Finkel A S and Redman S J Optimal voltage clamping with a single microelectrode Voltage and Patch Clamping with Microelectrodes Smith T G Lecar H Redman S J Gage P W eds Williams Wilkins Baltimo...

Page 194: ...od for perforated patch recordings using nystatin fluorescein mixture Jap J Physiol 43 267 273 1993 Microelectrodes Purves R D Microelectrode Methods for Intracellular Recording and Iontophoresis Acad...

Page 195: ...mp recordings from neurons of the mammalian central nervous system Pfl gers Arch 414 600 612 1989 Finkel A S Progress in instrumentation technology for recording from single channel and small cells Mo...

Page 196: ...nels detected with a new patch clamp configuration Nature 348 545 547 1990 Sakmann B and Neher E Single Channel Recording Plenum Press New York 1983 Wilson W A and Goldner M M Voltage clamping with a...

Page 197: ...olecular Devices will be pleased to supply them to you However we caution you that the Axoclamp 900A is a sophisticated instrument and that service should only be undertaken by talented electronics ex...

Page 198: ...uit diagrams and parts lists for the Axoclamp 900A I agree that I will only use the circuit diagrams and parts lists for service of the Axoclamp 900A I will not use them to create equivalent or compet...

Page 199: ...900A can be directly connected to all international supply voltages The input range is from 100 to 240 V No range switching is required Alternatively the instrument can be powered by a DC voltage of...

Page 200: ...should touch a grounded metal object immediately before touching the headstage Shipping the Axoclamp 900A The Axoclamp 900A is a solidly built instrument designed to survive shipping around the world...

Page 201: ...tourner le support du fusible dans le sens oppos des aiguilles d une montre 3 Remplacer le fusible par un fusible de m me valeur 4 Rebrancher la prise de courant Installation du mat riel et s curit 1...

Page 202: ...elui ci n est plus disponible il est recommand d envelopper soigneusement le Axoclamp 900A dans au moins trois inches 75 mm de mousse ou de feuilles d emballage bulles Le Axoclamp 900A ainsi prot g de...

Page 203: ...ung das Netzkabel ausschalten Vor dem Auswechseln der Sicherung den Grund f r ihren Ausfall untersuchen Schritte zum Auswechseln der Sicherung 1 Das Netzkabel ausschalten 2 Die Fassung der Sicherung m...

Page 204: ...ent das beim weltweiten Versand keinen Schaden nehmen sollte Um jedoch Versandsch den zu verhindern mu der Axoclamp 900A ordnungsgem verpackt werden Im allgemeinen l t sich der Axoclamp 900A am besten...

Page 205: ...lla Para cambiar el fusible 1 Desconecte el cord n el ctrico 2 Use un destornillador o un dispositivo similar para girar el portafusibles en sentido contrario al de las manecillas del reloj 3 Reemplac...

Page 206: ...Axoclamp 900A debe empacarse adecuadamente En general la mejor manera de empacar el Axoclamp 900A es en la caja original de f brica Si sta ya no se encuentra disponible le recomendamos que envuelva cu...

Page 207: ...the cell membrane from the outside surface to the inside surface Outward Current Current that flows from the inside to the outside surface of the cell Membrane Potential The potential inside the cell...

Page 208: ...e determined during subsequent data analysis Positive Current Current that flows out of the headstage into the electrode and out of the electrode tip into the cell Positive Negative Potential A positi...

Page 209: ...clamp dSEVC Discontinuous single electrode voltage clamp fs Sampling rate rate for switching from current passing to voltage recording in DCC and dSEVC modes G The average gain during dSEVC GT The in...

Page 210: ...with R0 R0 Current injection resistance Rs Resistance in series with membrane RMP Resting membrane potential Rm Rin Input resistance of cell membrane SEVC Single electrode voltage clamp TA Ambient tem...

Page 211: ...btracting Vb 68 Minimizing 66 Blank Activate 10 Blanking 110 Bridge Balance 87 115 116 118 119 141 144 Buzz 87 119 120 Capacitance Neutralization 64 83 86 87 118 119 142 143 144 Chamber Design 59 Chas...

Page 212: ...Leakage Current 77 Input Resistance 78 Interchangeability 77 VG 9A 70 High Voltage Current Clamp HVIC Mode 2 55 Holder Pipette Use 22 142 144 185 191 Holders 78 Ag AgCl Pellets 80 Cleaning 81 Design...

Page 213: ...l Test 11 110 132 153 Select Device 154 Setup 25 26 Sharp Microelectrode Recording 82 Signal Ground 10 Slow Current Injection 156 Soft Panel Configure 120 Space Clamp 164 Sync Output 11 133 Telegraphs...

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