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Axon MultiClamp 700A Theory And Operation

Computer-controlled microelectrode amplifier
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MultiClamp 700A
COMPUTER-CONTROLLED
MICROELECTRODE AMPLIFIER
Theory and Operation
Part Number 2500-129 Rev D November 2001 Printed in USA
Copyright 2000, 2001 Axon Instruments, Inc.
No part of this manual may be reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording, or otherwise, without written permission from Axon Instruments, Inc.
QUESTIONS? See www.moldev.com for how to contact Axon Instruments, Inc.

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  • Page 1 No part of this manual may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from Axon Instruments, Inc. QUESTIONS? See www.moldev.com for how to contact Axon Instruments, Inc.
  • Page 3 COPYRIGHT THE CIRCUITS AND INFORMATION IN THIS MANUAL ARE COPYRIGHTED AND MUST NOT BE REPRODUCED IN ANY FORM WHATSOEVER WITHOUT WRITTEN PERMISSION FROM AXON INSTRUMENTS, INC. VERIFICATION THIS INSTRUMENT IS EXTENSIVELY TESTED AND THOROUGHLY CALIBRATED BEFORE LEAVING THE FACTORY. NEVERTHELESS,...

  • Page 4: Declaration Of Conformity

    EN 50082-1: 1997 Safety: EN 61010-1: 2001 I, the undersigned, hereby declare that the equipment specified above conforms to the above Directives and Standards. Authorized Signature and Date: (Signature on file) MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 5 25 pin D-sub connector marked Shipping the MultiClamp 700A WARNING "Headstage". The MultiClamp 700A is a solidly IF THIS EQUIPMENT IS USED IN A Use: Do not operate this built instrument designed to survive MANNER NOT SPECIFIED BY THE equipment with covers or shipping around the world.
  • Page 6 IEC fourni pour brancher “headstage”, ceci risque de déranger son l'appareil a une prise de courant équilibre thermique. comprenant UNE TERRE. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 7 Alkohol oder vorsichtigem Abtupfen (FRAGILE) und einem Pfeil, der auf die Netzspannung mit entionisiertem Wasser ist eine Oberseite des Kartons weist, zu Der MultiClamp 700A kann direkt an alle wirksame Reinigung möglich. Die kennzeichnen. internationalen Netzspannungen Benutzung von Freon ist nach Sollte der Karton vom Spediteur angeschlossen werden.
  • Page 8 Suministro de corriente y conexión a que esté en contacto con tierra, antes de tierra: Use el cordón eléctrico IEC tocar el “headstage”. incluido para conectar el instrumento MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 9: Table Of Contents

    Test the Noise ......................9 Calibration ......................10 Getting Help in the MultiClamp Commander .............11 CHAPTER 3: USING THE MULTICLAMP 700A – TUTORIALS ....13 Check List........................13 Tutorial 1 – Electrode in the Bath: Voltage Clamp ..........14 Tutorial 2 – Electrode in the Bath: Current Clamp..........17 Tutorial 3 –...
  • Page 10 Bath Headstage and Electrodes ................60 Bridge Balance ......................63 Buzz.........................66 Capacitance Compensation..................67 Capacitance Neutralization..................73 Clear ........................76 Electrochemistry......................77 External Command Inputs ..................79 Feedback Resistor....................81 Filters........................83 Grounding and Hum ....................87 Headstage ........................89 Help .........................94 Holders ........................95 MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 11 Table of Contents • ix Input/Output Connections ..................99 Leak Subtraction....................102 Mode........................104 Model Cell ......................105 Noise........................107 Oscilloscope Triggering ..................110 Output Zero ......................111 Overload ........................112 Polarity Conventions .....................113 Power Supply......................115 Select Device ......................116 Series Resistance Compensation ................117 SoftPanel Configuration ..................130 Status ........................131 Zap .........................132 CHAPTER 6: TROUBLESHOOTING ..............133 CHAPTER 7: SPECIFICATIONS ...............135...

  • Page 13: Chapter 1: Introduction

    The design of the MultiClamp 700A allows it to support one or two headstages (CV- 7A), each of which contains circuitry that is virtually equivalent to an Axopatch-1D and the “bridge”...
  • Page 14 Scaling Factors and recording Mode. The Commander interface is completely independent of other software. Thus, the MultiClamp 700A can be used with any data acquisition package. It is, of course, compatible with all Digidata series digitizers and with pCLAMP 7 (and above) software.

  • Page 15: Chapter 2: Installation And Basic Operation

    Use the enclosed shipping list to verify that all parts have been received. For the initial checkout, the MultiClamp 700A should be situated on a bench top away from other equipment. Do not install it in a rack until the checkout is complete.

  • Page 16: Installing Hardware

    1. Connect one end of the RS232 “Null Modem” cable to the RS232 IN connector on the MultiClamp 700A main unit and the other end to a free serial port on your PC. Use the DB9 to DB25 adapter if the computer port is a 9-pin type.
  • Page 17 Installation and Basic Operation • 5 3. Ensure that the MultiClamp Hardware option is selected. Set Serial Port: to the number of the port to which the serial cable is connected (COM1 - 4). If you are not sure about the port number, continue to the next step. 4.

  • Page 18: Functional Checkout

    Tutorials (see page 13) and Calibration procedure (see page 10) be followed for maximum benefit. Communication with the MultiClamp 700A 1. Check that the STATUS LED on the front of the MultiClamp 700A is flashing. This indicates that the MultiClamp Commander is polling the MultiClamp 700A, updating its meter displays.
  • Page 19 Installation and Basic Operation • 7 1. Glider control • Position the cursor over the parameter field to the right of Holding, noting that the cursor changes to a vertical double-headed arrow ( ). Hold down the left mouse button and drag the mouse up and down; the holding potential changes in 1 mV steps.

  • Page 20: Toolbar Buttons In The Multiclamp Commander

    Save Settings toolbar button. Use the Save Settings dialog to enter a file name and directory. The file name is given the extension MCC (for MultiClamp Commander file). MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 21: Test The Noise

    All electronic equipment generates some amount of thermal noise. Follow these steps to measure the intrinsic current noise (“Irms”, or the root-mean-square of the current noise) of the MultiClamp 700A: 1. Leave the CV-7A headstage in an “open circuit” configuration (i.e., nothing should be attached to the input of the CV-7A).

  • Page 22: Calibration

    • Procedure for checking the calibration of the MultiClamp 700A. The steps below provide a quick check of the calibration of the MultiClamp 700A. It is assumed that appropriate shielding (as described in “Test the Noise”, above) will be used during these tests.
  • Page 23 Installation and Basic Operation • 11 3. Check Seal Test and set the amplitude to 100 mV, and frequency to 50 Hz. 4. Set the Scaled Output filter to 30 kHz. 5. Press Auto Cp Fast to remove the electrode capacitance transient. The waveform should be a square wave with about 100% overshoot settling to the baseline in about 1 to 2 ms.

  • Page 24: Getting Help In The Multiclamp Commander

    Commander, because this information is better provided in an interactive way using the On-line Help. Rather, the purpose of this manual is to provide tutorials and detailed information about the design and operation of the MultiClamp 700A amplifier as a whole. Therefore, the On-line Help and this manual complement each other.

  • Page 25: Chapter 3: Using The Multiclamp 700A - Tutorials

    The purpose of this chapter is to lead the user through the basics of patch clamping and ‘sharp’ microelectrode recording, using the PATCH-1U model cell that comes with the MultiClamp 700A. The tutorials are designed to illustrate the operation of the MultiClamp 700A and the MultiClamp Commander. Although this chapter is...

  • Page 26: Tutorial 1 - Electrode In The Bath: Voltage Clamp

    4 mm Signal Ground plug on the rear of the MultiClamp 700A. 4. Oscilloscope, connected to the MultiClamp 700A by means of a BNC cable, with which to monitor the output. The tutorials will assume an oscilloscope is being used.
  • Page 27 4. Press the Pipette Offset button while looking at the oscilloscope. Figure 2.2-3 After making a brief series of steps (due to the MultiClamp 700A’s algorithm for finding the offset) the Membrane Current is zeroed. Note also that the Pipette Offset button is grayed out and the lock closes.
  • Page 28 SYNC output on the rear of the MultiClamp 700A to the External Trigger input on the oscilloscope.) The amplitude of the Seal Test pulse is 10 mV. The amplitude of the Membrane Current output pulse is 0.5 V, which corresponds to 1 nA at the...

  • Page 29: Tutorial 2 - Electrode In The Bath: Current Clamp

    However, the principles illustrated are the same. 1. Set up the MultiClamp 700A and the MultiClamp Commander as in Steps 1-3 of Tutorial 1. 2. Under Channel 1 Mode: press the button labeled IC. The tab labeled I-Clamp 1 will select, and the Current Clamp light on the front panel of the MultiClamp 700A unit will illuminate.

  • Page 30: Tutorial 3 - Giga Seal Configuration

    Tutorial 1, Step 6. Tutorial 3 – Giga Seal Configuration 1. Set up the MultiClamp 700A and the MultiClamp Commander as in Steps 1-3 of Tutorial 1, except that the PATCH connector on the model cell should be plugged into the headstage of the MultiClamp 700A.
  • Page 31 Tutorials • 19 This opens the Options panel. Select the Gains tab. You will note that the default Feedback Resistor under Channel 1 Voltage Clamp is 500 MΩ. Increasing the size of the feedback resistor, which is located in the headstage, increases the gain of the headstage.
  • Page 32 • Scope is used to filter the signal provided by the SCOPE BNC on the front panel of the MultiClamp 700A. In the default configuration, this BNC simply duplicates the signal available at the SCALED OUTPUT BNC. However, in some circumstances you may wish to filter the SCOPE signal, being viewed on an oscilloscope, more heavily than the SCALED OUTPUT signal being sent to a computer.
  • Page 33 Tutorials • 21 Figure 2.13 The transients result from the charging of the 5 pF capacitance of the model cell, which simulates the capacitance of a patch electrode. In a real experiment these transients are undesirable because they may saturate the amplifier, leading to distortions in the measured currents.
  • Page 34 11. Now that the capacitance transients are compensated, it will be possible to increase the amplitude of the Seal Test pulse without overloading the MultiClamp 700A. Set the Seal Test amplitude to 100 mV by placing the cursor over the display (10 mV), double clicking and typing 100 <Enter>.

  • Page 35: Tutorial 4 - Whole-Cell Configuration: Voltage Clamp

    1. Reset to Program Defaults and set Seal Test frequency to 200 Hz. Plug the CELL connector on the model cell into the headstage of the MultiClamp 700A. 2. Check the Seal Test checkbox; a train of ~0.5 Volt transients decaying over ~1 ms will appear on the Scaled Output trace.
  • Page 36 After imposing a series of voltage steps on the model cell, the algorithm should converge on about 30 pF and 10 MΩ. In real experiments it may be necessary to make manual adjustments for optimum cancellation of the slow transient. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 37 The residual step, due to current flow through the “input resistance” of the model cell, can be canceled using the Leak Subtraction feature of the MultiClamp 700A. This subtracts from Scaled Output a current that is scaled linearly from the voltage command.
  • Page 38 Chapter 5. SERIES RESISTANCE COMPENSATION 9. The MultiClamp 700A is designed to be used with an external pulse generator or computer to provide voltage-clamp (and current-clamp) command steps. However, the Pulse button in the MultiClamp Commander allows you to apply simple, on-off steps with a selectable amplitude and duration.

  • Page 39: Tutorial 5 - Whole-Cell Configuration: Current Clamp

    Tutorials • 27 Tutorial 5 – Whole-Cell Configuration: Current Clamp 1. Set up the MultiClamp 700A and the MultiClamp Commander as in Step 1 of Tutorial 4. 2. Under Channel 1 Mode: press the button labeled IC. The tab labeled I-Clamp 1 appears, the Current Clamp light on the front panel of the MultiClamp 700A unit illuminates, and Scaled Output displays Membrane Potential.
  • Page 40 To the left of Bridge Balance is the Output Zero button. This works exactly like the corresponding button in voltage clamp, removing constant DC offsets. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 41 Tutorials • 29 6. In current-clamp mode the stray electrode capacitance can cause additional errors, acting to filter the membrane potential signal. This error can be reduced by using electronic compensation of the pipette capacitance. While holding down the Ctrl key to magnify mouse movement, use glider control to increase the Pipette Capacitance Neutralization (pF) value while monitoring Scaled Output on the oscilloscope.
  • Page 42 0 mV when I=0 is pressed. In a real cell the Membrane Potential would return to the resting potential of the cell. See IMPALING CELLS Chapter 4 for detailed information on current clamp experiments with real cells. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 43: Chapter 4: A Practical Guide To Electrophysiological Recording Using The Multiclamp 700A

    Recording Using the MultiClamp 700A The purpose of this chapter is to provide practical advice on patch clamping and sharp microelectrode recording, both of which are possible using the MultiClamp 700A. It includes both tutorial-style guidance and technical details for reference. This...

  • Page 44: General Advice

    For this reason, it is important to use a good, drift-free micromanipulator for the electrode, and to secure the tissue or cells in the chamber so they cannot move MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 45 Guide to Electrophysiological Recording • 33 very far. Tissue slices are commonly held in place in the chamber by a weighted “net” or “grid” of fine threads. A grid is easily made as follows. Bend a piece of 0.2-0.4 mm diameter platinum wire into a ring small enough to fit in the bottom of your chamber, then flatten the wire in a vise.

  • Page 46: Interfacing A Computer

    If you use both headstages on the MultiClamp 700A (e.g. for making simultaneous recordings from pairs of cells) you may wonder whether one or both headstage ground sockets need to be connected to the bath electrode. We have found empirically that the noise in the recordings depends on which headstage is grounded and what mode it is in (V-Clamp or I-Clamp).

  • Page 47: Patch Clamping

    Digital computers can generate considerable electrical noise, both via the power ground and via radiative interference from the monitor. For optimal noise performance of the MultiClamp 700A, careful attention should be paid to the placement of the computer. For example, the monitor should not be placed immediately above or below the MultiClamp 700A in the instrument rack.

  • Page 48: Forming A Gigaseal

    Techniques”, below. Forming a Gigaseal Start with the MultiClamp 700A in voltage clamp mode (VC). Fill a patch pipette with internal solution and secure it firmly in the pipette holder (fill the patch pipette with external solution if cell-attached recording is the goal). Be sure to support the...
  • Page 49 Guide to Electrophysiological Recording • 37 headstage with your other hand so that the micromanipulator will not have to absorb your force. Apply about 30 mbar of positive pressure to the holder tubing, then lower the pipette tip into the bath. Any voltage offset between the bath electrode and the patch electrode will show up as a non-zero tracking voltage on the I (nA) meter of the MultiClamp Commander.
  • Page 50 The best gigaseals are those that form nearly instantaneously. If a seal does not form within about a minute, continued suction is usually pointless. It is best to change electrodes and try again. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 51: Whole-Cell Voltage Clamp Recording

    If you intend to apply voltage steps to the patch, you may wish to use the Leak Subtraction feature of the MultiClamp 700A. This subtracts a scaled (divided by the resistance) version of the command pulse from the membrane current signal, and is particularly intended for use at high gains where the interesting single-channel currents are sitting on top of a leak current that may saturate the digitizing interface.
  • Page 52 The MultiClamp 700A contains a Zap circuit to aid in breaking into the cell. This circuit delivers a pulse of 1 V DC to the patch for variable durations ranging from 0.1 to 10 ms.
  • Page 53 Chapter 5, SERIES RESISTANCE COMPENSATION The Leak Subtraction feature of the MultiClamp 700A allows you to subtract linear leak currents from the membrane current traces. Generally speaking it is not a good idea to do this in the whole-cell configuration, because whole cells may contain background currents that have some dependence on voltage.

  • Page 54: Perforated-Patch Recording

    Figure 3.3. The Membrane Test feature of Clampex (v. 7 and higher) allows graphically monitoring the gradual rise in capacitance (and decrease in Rs) as pores are formed in the patch membrane. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 55: Low Noise Techniques

    Figure 3.3. Going whole-cell: capacity transients observed during amphotericin partitioning. Low Noise Techniques The MultiClamp 700A is capable of producing stable, low-noise recordings. To realize this performance the user must pay close attention to other sources of noise. This is because the total rms noise of a patch clamp recording is the square root of the sum of the individual squared rms noise sources.
  • Page 56 The deviation from 90° is the "loss factor". The loss factor is related to the power dissipated in the dielectric. e.g. Since energy is lost in the dielectric, dielectrics ( , glasses) are commonly referred to as "lossy". MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 57 Guide to Electrophysiological Recording • 45 any glass may potentially modify channel currents, one must be aware of this fact and control for it regardless of the glass one uses. We recommend two glasses for noise-critical work: Corning #7052 and quartz. Both have been successfully sealed to many different cell types.
  • Page 58 Therefore, for the most demanding low-noise applications it is recommended that an Axopatch 200B is used. Electrode Holder The holders supplied with the MultiClamp 700A are made of polycarbonate. Polycarbonate was experimentally found to produce the lowest noise among ten substances tested. It was only slightly better than polyethylene, polypropylene, and Teflon, but was much better than nylon, Plexiglass, and Delrin.

  • Page 59: Sharp Microelectrode Recording

    Signal Generator One last potential noise source to consider is the noise in the signal generator that provides the command. In the MultiClamp 700A we have succeeded in minimizing this noise by heavily attenuating the external command. However, it is possible for this noise source to be significant, particularly if the command signal comes from a D/A converter.

  • Page 60: Sharp Microelectrode Or Patch Electrode

    Microelectrode Properties Users of sharp microelectrodes spend far more time than patch clampers worrying about the properties of their electrodes. This is because the higher resistance of sharp MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 61 Guide to Electrophysiological Recording • 49 microelectrodes may introduce a number of undesirable properties. For best results, the microelectrode voltage must settle rapidly after a current pulse, and the microelectrode must be able to pass current without large changes in resistance. The important factors that need to be considered are discussed below.
  • Page 62 Another method is to coat the microelectrode with Sylgard #184 or Q-dope (model airplane glue). The selected material should be painted onto the electrode to within 100 µm of the tip. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 63 Guide to Electrophysiological Recording • 51 Tip Potentials During the passage of current, a slowly changing voltage may be generated at the tip of a microelectrode. Changes in this tip potential are indistinguishable from changes in the membrane potential and can therefore be a serious source of error.

  • Page 64: Filling Solutions

    Impaling Cells Start with the MultiClamp 700A in IC mode (I-Clamp). Fill a microelectrode with internal solution and secure it firmly in the pipette holder. Be sure to support the headstage with your other hand so that the micromanipulator will not have to absorb your force.
  • Page 65 Guide to Electrophysiological Recording • 53 Check the Tuning checkbox and observe the Scaled Output: Membrane Potential on a scope. Move the electrode tip close to where cells are likely to be encountered, and then increase Pipette Capacitance Neutralization in the MultiClamp Commander to give the fastest step response.

  • Page 67: Chapter 5: Reference Section

    MultiClamp 700A. This chapter provides details of the theory and operation of the MultiClamp 700A, beyond what is available in the On-line Help. The information in this section is gathered under a number of broad topics, arranged in alphabetical order.

  • Page 68: Audio Monitor

    DC signal, e.g. the membrane potential. The default setting for the VCO is 2200 Hz at 0 V ranging to 300 Hz at -100 mV. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 69 Reference Section • 57 Audio output can be monitored by making connections to the MultiClamp 700A in one of three different ways: 1. Connect the rear panel AUDIO OUTPUT to the Line IN connector of your computer sound card. This allows the MultiClamp 700A to use the computer’s speaker.
  • Page 70 2. Connect headphones or remote powered speakers to the front panel PHONES output or the rear panel AUDIO OUTPUT. This allows dedicated use of the headphones or external speakers by the MultiClamp 700A. Figure 4.2. Possible Audio configuration #2. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 71 3. Connect the Line OUT of your computer sound card to the rear panel AUDIO INPUT of the MultiClamp 700A, and the rear panel AUDIO OUTPUT to external powered speakers. This is the same as option 2, except that now the MultiClamp 700A audio output is mixed with the computer’s audio output to external speakers.

  • Page 72: Bath Headstage And Electrodes

    The Bath Headstage is used when recording from cells with a large conductance, in order to minimize errors due to current flow through the bath electrode. The VG-2 series Bath Headstage is optional hardware that can be used with the MultiClamp 700A for this purpose.
  • Page 73 This is achieved using a virtual-ground circuit, the bath headstage. The MultiClamp 700A is compatible with one of the following bath headstages from Axon Instruments: VG-2-x0.1 and VG-2A-x100. These headstages attach to the MultiClamp 700A via the rear-panel 15-pin D connector.
  • Page 74 SENSE electrode is equal to the potential at the positive input, i.e. 0 mV, irrespective of the voltage drop across R MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 75: Bridge Balance

    Reference Section • 63 Bridge Balance • Used to subtract voltage drops across the microelectrode when in I-Clamp mode. • Bridge balance is activated by pressing the button in the Bridge Balance box in the I-Clamp pane or by checking the checkbox and using manual glider control.
  • Page 76 Neutralization should also be set. (See Capacitance Neutralization.) Both settings can be monitored continuously through the experiment by injecting a small current step near the beginning of each data sweep. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 77 Reference Section • 65 It is recommended that Pipette Capacitance Neutralization be set at the same time as Bridge Balance, because both the electrode capacitance and the electrode resistance cause errors if left uncompensated. Also, it is easier to correctly balance the bridge when electrode capacitance is minimized, because the “break”...

  • Page 78: Buzz

    An appropriate duration can be found for most cells that is sufficiently long to allow penetration of the membrane but short enough that the cell is not damaged after penetration. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 79: Capacitance Compensation

    Reference Section • 67 Capacitance Compensation • Used to compensate electrode and cell capacitance when in V-Clamp mode. • Electrode capacitance is compensated using the controls in the V-Clamp pane. • Cell capacitance is compensated by checking the checkbox and using the associated controls in the V-Clamp pane.
  • Page 80 This takes longer to charge to its final value and is compensated by the C Slow controls. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 81 Furthermore, as in the case of the electrode capacitance transient, the whole-cell transient may saturate the circuitry of the MultiClamp 700A or downstream instruments if left uncompensated. Finally, whole- cell capacitance compensation is necessary for series resistance compensation. For all of these reasons, it is desirable to electronically compensate the capacitance of the cell.
  • Page 82 R to C2, the series resistance compensation circuitry can operate without causing the headstage input to saturate. (See also Chapter 5, ERIES ESISTANCE OMPENSATION MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 83 (The I trace in the figure was recorded using an oscilloscope probe connected to the internal circuitry). The Membrane Current and Command Potential outputs on the MultiClamp 700A would look like the I and V traces, respectively (Figure 4.8). It is easy to mistakenly think that the time course for charging the membrane is very fast but this is clearly not the case.
  • Page 84 This can be achieved by filtering the command stimulus before it is applied to the cell. This filtering can be done within the MultiClamp 700A. (See Chapter 5, EXTERNAL COMMAND INPUTS MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 85: Capacitance Neutralization

    MICROELECTRODE RECORDING • Electronically neutralize C The second approach has been implemented in the MultiClamp 700A in two ways. Primary Method for Neutralizing C A special technique is used in the CV-7A headstage to keep the contribution to from the input amplifier as small as possible. The technique is known as “bootstrapping”.
  • Page 86 Sometimes the overshoot is difficult to see. In this case, you may prefer to look at the “Scaled Output: Membrane Potential” trace at high gain on an oscilloscope, advancing the Pipette Capacitance Neutralization value until the trace becomes noisy and MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 87 Use of capacitance neutralization is less desirable than physically minimizing C since the neutralizing circuit adds noise to the voltage signal. This noise has been minimized in the CV-7A headstage of the MultiClamp 700A by using low-noise amplifiers and small injection capacitors, but it is still significant.

  • Page 88: Clear

    Whether to use a hyperpolarizing or depolarizing current depends on the preparation and must be determined by trial and error. Like Buzz, the mechanism for impalement is unknown. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 89: Electrochemistry

    For biological applications, the sensor is typically a carbon-fiber microelectrode. Examples of electro-active biological chemicals are dopamine and norepinephrine. The MultiClamp 700A, like the Axopatch 200B, can be used to measure the electrical signals generated by the presence of these chemicals.
  • Page 90 The temporal resolution is determined only by the response times of the sensor and the voltage clamp. Both cyclic voltammetry and amperometry can be performed by the MultiClamp 700A without modifications. Such modifications are necessary for some other Axon amplifiers because electrochemistry typically requires larger voltage commands than is usual for patch or intracellular recording.

  • Page 91: External Command Inputs

    External stimulus commands are supplied to the MultiClamp 700A via the COMMAND BNC on the front panel (one BNC for each Channel). Note that this is a DC-coupled input, so be sure that the external pulse generator is correctly calibrated so that zero volts really correspond to zero.
  • Page 92 Gains tab under Current Clamp.) Additivity of Commands All command stimuli applied by the MultiClamp 700A are additive. That is, the external command is algebraically added to Holding, Pulse and Seal Test or Tuning commands before the sum is applied to the cell.

  • Page 93: Feedback Resistor

    Note: V is limited to 10 V in the MultiClamp 700A, which in turn limits the maximum amount of current that can be injected throught the headstage resistor into the electrode. For example, with R = 500 MΩ, the maximum current that can be...
  • Page 94 Be aware that incompletely compensated capacitance transients, which are brief and often hard to see, may saturate before ionic currents. The OVERLOAD LED on the front panel of the MultiClamp 700A will assist you in judging when saturation has occurred.

  • Page 95: Filters

    Reference Section • 83 Filters • Low-pass and high-pass filters can be chosen to condition the Scaled Output and Scope outputs. The -3 dB frequency is selectable from a list in the Output Signals section of the main MultiClamp Commander window. •...
  • Page 96 The Bessel filter does not provide as sharp a roll- off as the Butterworth filter, but it is well behaved at sharp transitions in the signal, such as might occur at capacitance transients or single-channel current steps. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 97 Nyquist frequency. In practice, it is better to sample at two or more times the Nyquist frequency. Thus, f = 5f is commonly used. This means that, if the MultiClamp 700A filter is set at 5 kHz, your interface should be capable of digitizing at 25 kHz. Chapter 5...
  • Page 98 (See Chapter 5, EXTERNAL COMMAND INPUTS MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 99: Grounding And Hum

    • Place the MultiClamp 700A in the rack in a position where it will not absorb radiation from adjacent equipment. A grounded, thick sheet of steel placed between the MultiClamp 700A and the radiating equipment can effectively reduce induced hum.
  • Page 100 Such a procedure is too prone to accidental alteration. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 101: Headstage

    There are two types of I-V converters used in patch clamp headstages: capacitive feedback (used in the Axopatch 200B), and resistive feedback (used in the Axopatch-1D and in the MultiClamp 700A). The essential parts of a resistive-feedback headstage are shown in Figure 4.11.
  • Page 102 This means the voltage clamp of the electrode is fast. The RC filtering effect mentioned above applies only to the output of the I-V converter, which can therefore be subjected to post hoc boosting. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 103 Reference Section • 91 What is Clamped During Voltage Clamping? We were careful to state in the above discussion that it is only the back of the electrode that is voltage clamped, not the cell membrane. The voltage at the cell membrane may differ from that at the back of the electrode because of bandwidth and voltage errors due to uncompensated series resistance (R ).
  • Page 104 Figure 4.12. Voltage follower headstage. Note that V is limited to 10 V in the MultiClamp 700A, which in turn limits the maximum amount of current that can be injected throught the headstage resistor into the electrode. For example, with R = 500 MΩ, the maximum current that can be...
  • Page 105 Reference Section • 93 Cleaning Wipe the headstage connector with a damp cloth to clean salt spills. Avoid spilling liquids on the headstage. The Teflon input connector should be kept very clean. Effective cleaning can be done by spraying with alcohol or swabbing carefully with deionized water.

  • Page 106: Help

    Commander must have a web browser (Internet Explorer v. 4 or later, or equivalent). JavaScript is required. When the user clicks on the Help button, the browser will open automatically (if it is not already running) and the relevant page will appear. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 107: Holders

    (The electrode should always be inserted as far as it will go in the holder.) The holder mates with the threaded Teflon connector on U-type Axon headstages and is secured in place with a threaded collar. Figure 4.13. Exploded view of the HL-U holder.
  • Page 108 (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 sold as HLA-003. Figure 4.14. Ag/AgCl pellet assembly. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 109 For easy-to-use recipes see Microelectrode Methods for Intracellular Recording and Ionophoresis, by R.D. Purves, London: Academic Press, 1981, p. 51 or The Axon Guide. Foster City, CA: Axon Instruments, Inc., 1993, p. 83. Chapter 5...
  • Page 110 Use the HLR-U with the HL-U holder. HLB-U BNC-to-Axon adapter allows conventional BNC-type holders to be used with Axon U-type headstages. Use the HLB-U with all U-type CV and HS headstages. These headstages have a threaded white Teflon collet.

  • Page 111: Input/Output Connections

    Gains panel under the Options toolbar button. Outputs SCALED: This is intended to be the primary conditioned output of the MultiClamp 700A. Its identity is selected from the list in the Output Signals section of the main window of the MultiClamp Commander: •...
  • Page 112 AUDIO INPUT: This connector is used if you wish to mix the audio output of the MultiClamp 700A with the audio output of your PC. Connect the audio output of your PC’s sound card to the AUDIO INPUT socket and the MultiClamp’s AUDIO OUTPUT socket to the PC-powered speakers.
  • Page 113 SIGNAL GROUND: This 4 mm socket is the primary grounding point for the MultiClamp 700A. Ideally, it should be connected to a grounding bus to which is also connected the Faraday cage and the signal grounds of other instruments used in your experiment.

  • Page 114: Leak Subtraction

    1 GΩ seal resistance. The seal (leak) current during the step will be 100 pA. Because of this relatively large leak current, the gain of the MultiClamp 700A cannot be turned up very far without saturating the amplifier, but at a low gain setting the single-channel openings may not be resolved very well.
  • Page 115 Reference Section • 103 We recommend that Leak Subtraction be used with caution, because it assumes that is constant for all voltage steps. This may not be true if, for instance, the patch seal contains small channels or electrogenic transporters that do not produce discernible single-channel events.

  • Page 116: Mode

    • See also Headstage, Input/Output Connections. Switching between V-Clamp and I-Clamp modes in the MultiClamp 700A activates a switch between two distinct circuits in the CV-7A headstage. Voltage clamp is achieved with a current-voltage converter, whereas current clamp is achieved with a voltage follower.

  • Page 117: Model Cell

    Reference Section • 105 Model Cell • PATCH-1U model cell is a standard accessory provided with the MultiClamp 700A. It is useful for setting up, testing and doing the tutorials described in Chapter 3. The model cell is a small metal box with three connectors labeled BATH, CELL and PATCH, and an unlabeled 2 mm gold plug which connects to the 1 mm grounding plug on the rear of the CV-7A headstage.
  • Page 118 106 • Reference Section Figure 4.15. PATCH-1U model cell. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 119: Noise

    Reference Section • 107 Noise • Sources of instrument noise in the MultiClamp 700A. • See also Feedback Resistor, Filters, Grounding and Hum, Headstage, Power Supply, Series Resistance Compensation. Measurement of Noise Noise is reported in two different ways in this manual.
  • Page 120 I-Clamp: Voltage noise increases markedly with electrode capacitance and resistance. Thus, both should be minimized as much as possible. (See Chapter 4, SHARP MICROELECTRODE RECORDING MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 121 Reference Section • 109 Headstage Circuit V-Clamp: Current noise decreases as the value of the feedback resistor (R ) is increased. Thus, for minimum noise the largest R should be chosen, subject of course to range limitations. (See Chapter 5, FEEDBACK RESISTOR I-Clamp: Voltage noise decreases as the value of R is decreased, but R...

  • Page 122: Oscilloscope Triggering

    110 • Reference Section Oscilloscope Triggering • SYNC output on the rear panel of the MultiClamp 700A provides a signal for triggering an oscilloscope (or for triggering in Clampex). • See also Input/Output Connections. The signal available at this BNC connector is intended to be used as an External trigger for an oscilloscope when Seal Test (in V-Clamp), Tuning (in I-Clamp) or Pulse is activated.

  • Page 123: Output Zero

    Reference Section • 111 Output Zero • Subtracts the steady-state current offset (in VC mode) or voltage offset (in IC mode). • Activated by pressing the button in the Output Zero box, or by checking the checkbox and manually adjusting the value to the left of the button. •...

  • Page 124: Overload

    Overload • OVERLOAD LED on the front panel of the MultiClamp 700A warns when the signal presented at SCALED OUTPUT or SCOPE saturates (i.e. exceeds ±10.5 V longer than 10 µs) at any point in the internal circuitry of the amplifier.

  • Page 125: Polarity Conventions

    That is, the current is defined with respect to the direction of flow into or out of the headstage. Axon amplifiers do not have switches that reverse the current or the voltage command polarities. This prevents forgetting to move the...
  • Page 126 Membrane potential = V Inside-out Patch Positive current = inward membrane current Membrane potential = – V Cell-attached Patch Positive current = inward membrane current Membrane potential = V – V rest MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 127: Power Supply

    Changing the Fuse The MultiClamp 700A uses a 0.5 A, 250 V slow acting 5 x 20 mm fuse. Before changing the fuse investigate the reason for its failure. To change the fuse: 1. Disconnect the power cord.

  • Page 128: Select Device

    Device Number (0-9) of the MultiClamp 700A. The number of the computer’s serial port connected to the MultiClamp 700A sets the Serial Port number. The Device Number is set by a rotary switch on the rear panel of the MultiClamp 700A (default is 0).

  • Page 129: Series Resistance Compensation

    Reference Section • 117 Series Resistance Compensation • Theory and practice of compensating the series resistance in V-Clamp mode. • Adjusted using the controls in the V-Clamp pane. • See also Capacitance Compensation, Headstage. Introduction to R Compensation Series resistance (R ) is defined as the total resistance that is interposed between the circuitry of the headstage and the membrane of the cell.
  • Page 130 100 pA, the steady-state voltage error will be at most 10 MΩ x 100 pA = 1 mV which is probably insignificant. In this case you might think that R compensation is not necessary. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 131 Reference Section • 119 However, it should be remembered that dynamic voltage errors and bandwidth errors can still occur in the above example, because these depend on R and C and not on the size of the membrane current. Even if you are measuring only small membrane currents in a whole-cell recording, application of R compensation can greatly improve the fidelity of the voltage clamp.
  • Page 132 Figures 4.15-4.19. Whole cell responses using PATCH-1U model cell with R = 500 MΩ, Scaled Output gain = 10 and Seal Test = 100 mV. Figure 4.15. Uncompensated response (with saturating transients). Figure 4.16. After compensating transients. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 133 Reference Section • 121 Figure 4.17. After setting Prediction = 90%, Correction = 0%. Figure 4.18. After setting Prediction = 90%, Correction = 90%. Figure 4.19. After optimizing C and C to minimize transients. Chapter 5...
  • Page 134 In order to see the improvement brought about by R compensation, check and uncheck the R Compensation checkbox. A dramatic speeding-up of the Membrane Current should be apparent with the compensation correctly adjusted. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 135 Reference Section • 123 Theory of R Compensation The MultiClamp 700A uses a dual approach for R compensation, like the Axopatch 200 series of amplifiers. This provides superior correction and stability. For R compensation to function properly, whole cell compensation must have been adjusted and the Whole Cell checkbox must be checked.
  • Page 136 (1 - % Prediction /100); here this is 250 µs. More formally, the command potential with the Prediction signal included, V , can be expressed in terms of the command input, V , by: (1+sτ )/(1+sτ MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 137 Prediction circuit itself.) The command plus Prediction signal is attenuated at the headstage by a 10:1 voltage divider. Since the circuitry in the MultiClamp 700A main unit will saturate at about ±11-12 V, V is limited in absolute value to about 1.1 to...
  • Page 138 The percentage set by the Correction potentiometer refers to the R (MΩ) value under Whole Cell. For example, if this value is 10 MΩ, a 90% setting of the Correction control means MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 139 Reference Section • 127 that 9 MΩ of series resistance is compensated; the residual (uncompensated) series resistance in terms of Correction, R , is 1 MΩ. The Bandwidth setting under R Compensation gives the –3 dB cutoff frequency of a one-pole RC filter through which the Correction signal is passed prior to being summed with V .
  • Page 140 Correction control. In other patch clamps the issue of saturation would limit the amount of compensation used for ionic currents; this is not true in the MultiClamp 700A. On the other hand, in some cases it might be impossible to advance the Correction percentage beyond about 70% without causing instability.
  • Page 141 Reference Section • 129 The tendency to oscillate therefore depends on the relative magnitude of the electrode resistance to the electrode capacitance and the degree of compensation of the electrode capacitance. Thus, one should take care that C is well compensated as one advances correction. In addition, the tendency to oscillate can be reduced by limiting the bandwidth of the positive-feedback circuit.

  • Page 142: Softpanel Configuration

    Re-label the position with the appropriate function using a marking pen. (Sharpie® pens are appropriate on this special surface.) Figure 4.21 MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 143: Status

    Reference Section • 131 Status • STATUS LED on the front panel of the MultiClamp 700A indicates traffic on the serial cable. • See also Chapter 6, TROUBLESHOOTING The STATUS light illuminates whenever data is being transmitted on the serial cable that connects the MultiClamp 700A to the host computer.

  • Page 144: Zap

    Membrane Current. Sometimes it helps to apply steady suction while Zapping. Successful break-through is signaled by an increase in the current noise and by large whole-cell capacitance transients. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 145: Chapter 6: Troubleshooting

    Chapter 6 Troubleshooting It has been our experience at Axon Instruments that the majority of troubles reported to us have been caused by faulty equipment connected to our instruments. If you have a problem, please physically disconnect all instruments connected to the MultiClamp 700A except for the oscilloscope.
  • Page 146 Rechloride the Ag wire. Clean the holder and headstage connectors. If the problem cannot be resolved, please contact Axon Instruments for technical support 800-635-5577 or www.moldev.com/support MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 147: Chapter 7: Specifications

    Specifications • 135 Chapter 7 Specifications Unless otherwise specified, T = 20 C, 1 hr warm-up time. Main Unit Line Voltage 85 - 260V Line frequency 50 - 60 Hz Fuse 5 mm x 20 mm 0.5A slow Case 8.89 cm high x 48.26 cm x 30.48 cm deep (3.5˝ x 19˝ x 12˝ deep) rack mountable CV-7A Headstage Dimensions 4.06 x 8.38 x 2.03 cm (1.6˝...
  • Page 148 Rise time < 10 µs for load of 10 M on 50 M range (Output Filter bypassed). Rise time < 30 µs for load of 100 M on 500 M range. Rise time < 150 µs for load of 1 G on 5 G range. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 149 Specifications • 137 Test Signals Voltage Clamp The available test signals are Seal Test, Pulse and Zap. Seal Test and Pulse amplitudes are selectable from 0 to ±1 V at the electrode. Seal Test frequency is selectable from 2 to 1000 Hz. Pulse duration is selectable from 0.1 to 500 ms.
  • Page 150 When enabled in MultiClamp Commander software, 0 input selects I-Clamp mode and 5V input selects V-Clamp mode. Scope Filter Two pole Bessel low pass filter with four –3 dB cutoff frequencies (Hz): 1k, 3k, 10k, Bypass. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.
  • Page 151 Specifications • 139 Output Gain Post-filter gain of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000. Audio Monitor The Audio Monitor output can select Current, Voltage or Voltage x 100 for either Channel 1 or Channel 2. The selected signal is available for direct monitoring or via a voltage-to-frequency converter (VCO).

  • Page 153: References

    References • 141 References Armstrong, C.M. and Chow, R.H. Supercharging: a new method for improving patch- clamp performance. Biophys. J. 52:133-136, 1987. Ebihara, S., Shirato, K., Harata, N. and Akaike, N. Gramicidin-perforated patch recording: GABA response in mammalian neurones with intact intracellular chloride. J.
  • Page 154 Sherman-Gold, R. The Axon Guide for Electrophysiology & Biophysics Laboratory Techniques. Axon Instruments, Foster City, CA. 1993. Yawo, H. and Chuhma, N. An improved method for perforated patch recordings using nystatin-fluorescein mixture. Jap. J. Physiol. 43:267-273, 1993. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 155: Warranty And Repair Service

    The warranty is valid when the product is used for its intended purpose and does not cover products which have been modified without approval from Axon Instruments, or which have been damaged by abuse, accident or connection to incompatible equipment.
  • Page 156 Support Department. If it is determined your instrument must return to the factory for repair, the Technical Support Representative will issue a Return Merchandise Authorization (RMA) number. Our RMA Coordinator will contact you with specific instructions. MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

  • Page 157: Shipping

    If you need to ship the MultiClamp 700A to another location, or back to the factory, and you do not have a means to adequately package it, Axon Instruments can ship the proper packaging material to you for a small fee.

  • Page 158: Index

    Index • 149 Index Audio Monitor, 56, 57, 58, 59, 139 External Command Inputs, 67, 72, 79, 81, 83, 86, 99 Auto Filter, 79, 80, 84, 85, 86, 119, 122, 136, 138 Bridge Balance, 28, 53, 63, 64, 65, 73, 74, 111, Functional Checkout, 6 Leak Subtraction, 25, 39, 41, 102, 103, 111 Gain, 20, 100, 102, 112, 135, 138, 139...
  • Page 159 Prediction, 26, 121, 122, 124, 125, 126, 127, 128 Pulse, 26, 30, 79, 80, 101, 110, 137 Quick Select, 8, 9 Raw Output, 8 Reset to Program Defaults, 14, 23 MultiClamp 700A Theory and Operation, Copyright 2000, 2001 Axon Instruments, Inc.

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