Axon AutoMate Scientific MultiClamp 700B, Manual

Page 1: ...produced 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 Molecular Devices Corp QUESTIONS See Axon s Knowledge Base http support com axon 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 a x 5 10 6 6 5 3 9 7 5 e m a i l i...

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Page 3: ...before leaving the factory Nevertheless researchers should independently verify the basic accuracy of the controls using resistor capacitor models of their electrodes and cell membranes WARNING If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired DISCLAIMER This equipment is not intended to be used and should not be used i...

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Page 5: ...der 7 Toolbar Buttons in the MultiClamp 700B Commander 9 Test the Noise 10 Calibration 11 Getting Help in the MultiClamp 700B Commander 13 Chapter 3 Tutorials 15 Check List 15 Model Cell 16 Tutorial 1 Electrode in the Bath Voltage Clamp 17 Tutorial 2 Electrode in the Bath Current Clamp 19 Tutorial 3 Giga Seal Configuration 21 Tutorial 4 Whole Cell Configuration Voltage Clamp 26 Tutorial 5 Whole Ce...

Page 6: ... Noise Techniques 55 Sharp Microelectrode Recording 59 Sharp Microelectrode or Patch Electrode 60 Microelectrode Properties 60 Filling Solutions 64 Impaling Cells 64 Chapter 5 Reference Section 67 Audio Monitor 68 Bath Headstage and Electrodes 72 Rb Minimization 72 Use of a Bath Headstage 74 Bridge Balance 75 Bridge Balance in the Bath 76 Bridge Balance in the Cell 77 Buzz 78 Capacitance Compensat...

Page 7: ...xternal Command Sensitivity 89 Additivity of Commands 90 Command Filter Frequency 90 Feedback Resistor 91 V Clamp Mode 91 I Clamp Mode 93 Filters 93 3 dB Frequency 94 Types of Filters 95 Bessel Filter 95 Butterworth Filter 95 Choosing the Cutoff Frequency 95 High pass Filter 97 Command Filter Frequency 97 Grounding and Hum 97 Headstage 98 Voltage Clamp Circuit 99 Current Clamp Circuit 101 Mounting...

Page 8: ...pe Triggering 116 Output Zero 117 Overload 118 Polarity Conventions 118 Biological Polarity Conventions 118 MultiClamp Polarity Conventions 119 Polarity Summary for Different Recording Configurations 120 Power Supply 120 Supply Voltage Selection 121 Changing the Fuse 121 Glitches 121 Select Device 122 Series Resistance Compensation 122 Introduction to Rs Compensation 122 Is Rs Compensation Necessa...

Page 9: ... Cell Compensation with Correction 135 Setting Prediction and Correction Values 135 Oscillations 135 Limitations of Rs Compensation 136 SoftPanel Configuration 137 Status 138 Zap 138 Chapter 6 Troubleshooting 139 Chapter 7 Specifications 141 References 147 Technical Assistance 149 Warranty and Repair Service 151 Circuit Diagrams Request Form 155 Declaration of Conformity 157 Important Safety Infor...

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Page 11: ...ge contains a current to voltage converter for voltage patch clamp and a voltage follower for current clamp This allows the user to conveniently switch between low noise patch clamp recording and high speed current clamp recording Also an optional CV 7 headstage will allow bilayer recording The MultiClamp 700B is essentially an analog input output instrument similar to conventional amplifiers by A...

Page 12: ...device to control the MultiClamp 700B The SoftPanel is essentially a hardware extension of the MultiClamp 700B Commander software Knobs and buttons replace mouse or keyboard control For more information visit our website or call Axon Technical Support The MultiClamp 700B is a sophisticated instrument Experienced and inexperienced researchers alike are advised to read this manual thoroughly and to ...

Page 13: ...ned to the factory at a later date For the initial checkout the MultiClamp 700B should be situated on a bench top away from other equipment Do not install it in a rack until the checkout is complete Check List These installation and checkout procedures require the following 1 MultiClamp 700B main unit with power cord 2 CV 7 headstage s with PATCH 1U model cell s 3 USB A B type control cable 4 Mult...

Page 14: ...NECTED Note a small white cap covering one of the headstage input pin sockets and a corresponding missing pin on the headstage connector This is normal 3 Connect the power cable and turn on the MultiClamp 700B The front panel POWER light should illuminate as well as the VOLTAGE CLAMP light for each channel Windows operating system will automatically recognize the new USB hardware as a Human Interf...

Page 15: ...to check for a new download 3 Next you will see the Device Selection dialog Select MultiClamp Hardware then click the Scan button The amplifier Serial Number will be identified in the list when the instrument is successfully recognized Figure 2 1 If the program is unable to find a valid Serial Number check that the MultiClamp 700B is switched on and that the USB cable is connected properly 4 If yo...

Page 16: ...u step by step through the various recording configurations Communication with the MultiClamp 700B 1 Check that the STATUS light on the front of the MultiClamp 700B is flashing This indicates that the MultiClamp 700B Commander is polling the MultiClamp 700B updating its meter displays 2 Toggle the Channel 1 and Channel 2 Mode buttons switching repeatedly between Voltage Clamp VC and Current Clamp ...

Page 17: ...ng potential changes in 5 mV steps Press the Ctrl key while dragging the mouse the holding potential changes in 20 mV steps Position the cursor over the button with the black dot dual control to the right of Cp Fast noting that the cursor changes to crossed double headed arrows Holding down the left mouse button and dragging the mouse vertically changes the capacitance parameter pF while dragging ...

Page 18: ...4 Repeat for the Primary or Secondary Output field In this case a right click of the mouse will display a list of output signals Figure 2 5 Right clicking the mouse over most other 700B Commander glider fields will display a menu to select the sensitivity of the glider For example right click the mouse while over the Holding glider and you will see the following menu Figure 2 6 MultiClamp 700B The...

Page 19: ... Most other buttons are self explanatory with the possible exception of the Save Configuration Load Configuration and Quick Select buttons These buttons allow the user to store and retrieve parameter settings for the MultiClamp 700B Commander The Quick Select buttons can be assigned to a particular set of parameter settings to facilitate rapid loading or alternatively to run an executable command ...

Page 20: ...nic equipment generates some amount of thermal noise Follow these steps to measure the intrinsic MultiClamp 700B current noise Irms or the root mean square of the current noise 1 Leave the CV 7 headstage in an open circuit configuration i e nothing should be attached to the input of the CV 7 2 To reduce extraneous noise the CV 7 must be shielded This can be accomplished using aluminum foil which s...

Page 21: ... Resistor selected from the MultiClamp 700B Commander Options menu 6 If your MultiClamp has more than one CV 7 headstage repeat steps 1 5 for the second headstage Calibration The steps below provide a quick check of the calibration of the MultiClamp 700B It is assumed that appropriate shielding as described in Test the Noise above will be used during these tests 1 Connect an oscilloscope to the fr...

Page 22: ...he steady state response that settles to the baseline in about 1 to 2 ms The rise time to the peak of the overshoot should be about 50 µs The steady state amplitude following the transient should be 500 mVp p 50 mV 5 G Range 1 Change the feedback resistor to 5 G in the Options Gains menu 2 Press Auto Cp Fast 3 The step response should be 50 mVp p 5 mV 500 M Range 1 Press the Reset to Program Defau...

Page 23: ...ne Help which describes many of the functions of the MultiClamp 700B Commander This manual is designed to be used in conjunction with the On line Help This manual does not for example describe all the buttons and windows in MultiClamp 700B 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...

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Page 25: ...tware Although this chapter is directed primarily at inexperienced electrophysiologists it may also be useful for experienced researchers who desire a simple introduction to the features of the MultiClamp 700B We recommend that you perform the Tutorials in order to avoid confusion Check List These tutorials require the following 1 MultiClamp 700B main unit and at least one CV 7 headstage The tutor...

Page 26: ...circuits of resistors and capacitors designed to simulate three patch clamp recording conditions 1 Pipette in the bath Connector labeled BATH on the model cell 2 Gigaseal PATCH and 3 Whole cell CELL The circuit for each of these is as follows Also see MODEL CELL in Chapter 5 BATH 10 MΩ pipette resistor to ground PATCH 10 GΩ patch resistor to ground Approximately 5 pF stray capacitance to ground CE...

Page 27: ...d socket on the side of the model cell to the 1 mm gold socket on the rear of the CV 7 headstage using the short black wire provided with the model cell Shield the headstage and model cell with the aluminum foil or metal box Ground the shield by connecting using an alligator clip to the 1 mm plug inserted previously into the rear socket of the CV 7 3 Connect a BNC cable from the Channel 1 Primary ...

Page 28: ... 3 3 A repetitive pulse appears on the Primary output signal The trace can be triggered on the oscilloscope screen by making a connection from the SYNC output on the rear of the MultiClamp 700B to the External Trigger input on the oscilloscope See Options General tab 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 ...

Page 29: ...amplitude and frequency by using the glider control with the mouse See SETTING PARAMETERS IN THE MULTICLAMP 700B COMMANDER in Chapter 6 Figure 3 6 Note how the Primary Output signal changes on the oscilloscope as the test pulse parameters are changed Tutorial 2 Electrode in the Bath Current Clamp Note that the model cell used in this tutorial is designed to simulate a patch pipette rather than a t...

Page 30: ...Primary Output signal changes on the oscilloscope 4 Check the Tuning checkbox Figure 3 8 A repetitive pulse appears on the Membrane Potential output The amplitude of the Tuning pulse is 1 nA The amplitude of the Membrane Potential output pulse is 100 mV which corresponds to 10 mV at the default gain of 10 V mV Therefore the resistance of the model electrode is calculated from Ohm s Law to be R V I...

Page 31: ...t the Channel 1 Primary Output after turning up the vertical gain on the oscilloscope The noise on Primary Output should be about 5 mV peak to peak p p which corresponds to 10 pA p p at the default scale factor of 0 5V nA 10 pA is too noisy for most single channel recording 3 Press the Options toolbar button at the top of the MultiClamp 700B Commander Figure 3 9 This opens the Options panel Select...

Page 32: ...oamps To clearly see small currents it is necessary to filter the Primary Output 5 Locate the Primary Output section in the main window of the MultiClamp 700B Commander and position the mouse cursor over Bessel 10 kHz Using the glider control see Chapter 2 examine the effect of filtering the Primary Output Figure 3 11 Note that with a filter setting of 2 kHz the peak to peak noise on Primary Outpu...

Page 33: ...C allows you to send the Primary Output through a high pass filter This may be desirable if you wish to remove a DC offset or low frequency component in the signal output Scope is used to filter the signal provided by the SCOPE BNC on the front panel of the MultiClamp 700B In the default configuration this BNC simply duplicates the signal available at the Primary Output BNC However in some circums...

Page 34: ...he SYNC output of the MultiClamp 700B as described in Tutorial 1 Figure 3 12 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 They can be eliminated by using the Cp Fast an...

Page 35: ...me constant of the transients on the oscilloscope With Cp Fast capacitance set to about 5 pF the transients should be minimized 9 An alternative way to cancel the transients is by pressing the Auto button opposite Cp Fast The algorithm should find optimum values of about 5 pF and 1 µs In experiments with real cells you may need to make manual fine adjustments for optimal cancellation 10 Sometimes ...

Page 36: ...itudes of about 5 mV Figure 3 14 With the Primary Output Membrane Current gain set at 0 5 V nA this is equivalent to 10 pA Hence the resistance of the model patch is calculated from Ohm s Law to be R V I 100 mV 10 pA 10 GΩ Alternatively check the Resistance checkbox under the Channel 1 meters Tutorial 4 Whole Cell Configuration Voltage Clamp 1 Reset to Program Defaults and set Seal Test frequency ...

Page 37: ...he capacitance of the simulated cell 33 pF Following the 10 mV Seal Test step the transients decay to a plateau of 10 mV equivalent to a current of 20 pA This yields a resistance of 10 mV 20 pA 500 MΩ which is the input resistance of the model cell This can also be found by checking the Resistance checkbox under the meters 3 In a real cell the holding potential would have been set prior to going t...

Page 38: ...ed This can be removed by again pressing the Cp Fast Auto button 5 An alternative way to cancel the slow transient is by pressing the Auto button Try this after first using glider control to set the pF and MΩ values to wrong values such as 100 pF and 100 MΩ After imposing a series of voltage steps on the model cell the algorithm should converge on about 33 pF and 10 MΩ In real experiments it may b...

Page 39: ...e with Leak Subtraction the step due to Seal Test is not subtracted 7 The series resistance Rs which typically originates near the tip of the recording electrode can be thought of as an unwanted resistance that is interposed between the headstage circuitry and the membrane of the cell Since Rs can cause serious errors in voltage clamp mode it needs to be reduced as much as possible This can be don...

Page 40: ... Prediction and Correction that provides maximal compensation without oscillation Full details are given in SERIES RESISTANCE COMPENSATION in Chapter 5 9 The MultiClamp 700B 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 700B Commander allows you to apply simple on off steps wit...

Page 41: ...ront panel of the MultiClamp 700B unit illuminates and Primary Output displays Membrane Potential 4 Check the box next to Holding and using glider control vary the holding current pA while viewing the Primary Output signal on the oscilloscope and on the MultiClamp 700B Commander voltage meter The model membrane potential varies smoothly with Holding current Figure 3 21 5 Switch off Holding and che...

Page 42: ...introduce errors to current clamp recordings and needs to be compensated electronically In current clamp mode Rs is compensated using Bridge Balance Figure 3 24 Check the Bridge Balance checkbox and using glider control vary the MΩ value until the step is eliminated Alternatively press the Auto Bridge Balance button for automatic adjustment The electrode resistance of the model cell is 10 MΩ but i...

Page 43: ...on 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 Primary Output on the oscilloscope Figure 3 25 Note that as you increase the value beyond about 3 pF damped oscillations start to appear at the beginning of each sawtooth Figure 3 26 If you go further still full scal...

Page 44: ... disabling or alternatively reducing Capacitance Neutralization In a similar manner Series Resistance compensation can be disabled or reduced in Voltage Clamp Check the box beneath Pipette Capacitance Neutralization labeled Disable if oscillation detected Figure 3 27 9 Now increase the Pipette Capacitance Neutralization until you reach a value that evokes full scale oscillations The automatic prot...

Page 45: ...Capacitance Neutralization in IC mode Neutralization will be iteratively reduced by 1 pF steps until oscillations are no longer detected Note also that this menu applies a similar reducing effect to Series Resistance compensation if oscillations are detected in VC mode 11 Check on the Reduce Rs Compensation radio button in the Options Auto menu Close this menu then repeat step 8 above to evoke osc...

Page 46: ...ivate the slow current injection feature and monitor the Primary Output Membrane Potential on the oscilloscope You should note a voltage deflection from 0 V to 1 V since the scale factor is 10 mV mV The deflection will require approximately 500 ms to reach steady state Figure 3 31 The time required to reach the selected voltage depends upon the feedback resistor and headstage load See the MultiCla...

Page 47: ... are disconnected With the model cell the Primary Output Membrane Potential signal returns to near 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 in Chapter 4 for detailed information on current clamp experiments with real cells Tutorial 6 Whole Cell Configuration Automatic Mode Switching 1 Set up MultiClamp 700B ...

Page 48: ...or 100 pA 2 Hz then check box to activate 4 Click on the Options Auto tab In the Switch to voltage clamp section click the radio button for On positive to negative Vm threshold crossing Set Delay change to voltage clamp 0 ms and Membrane Potential Vm threshold 20 mV Next click the radio button for Return to current clamp After and set this value to 500 ms Close the Options menu to return to the ma...

Page 49: ...ent step Figure 3 35 6 Now check the Auto checkbox next to the Mode buttons Note that the VC I 0 and IC buttons are now greyed out since they are under automatic control Figure 3 36 7 Monitor the Primary Output on the oscilloscope You should now observe the following events see figure below a A switch from IC to VC when the negative going deflection of the membrane potential reaches 20 mV Chapter ...

Page 50: ... SYNC output monitoring Mode Auto ON Switch to VC at 20 mV Figure 3 37 8 The lower trace in the figure above shows a recording from the SYNC output on the rear of the MultiClamp 700B In the Options General tab select Mode on channel 1 to follow the mode switching event A deflection to 5 V indicates VC mode while 0 V is the output during IC mode 9 Note During a real experiment if you are using an e...

Page 51: ...ch settings To disable the external commands go to the Options Gains tab and click the OFF radio button in the VC and IC External Command Sensitivity sections Figure 3 38 10 Experiment with different settings Threshold crossing Delay Vm and Return in the Options Auto tab in order to appreciate the flexibility of this automatic Mode switching feature Chapter 3 ...

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Page 53: ... This information has been distilled from textbooks on the subject see References at the end of this manual and from experienced 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 provisional until they have been tested in your own circumstances This chapter has been d...

Page 54: ...ices have unusually high metabolic rates and require fast perfusion with high O2 solution to remain viable Other cells e g neurons in dissociated cell culture may not need any perfusion or bubbling at all Because the health of the cells is the single most important factor in determining the success of your experiments it is worth spending some time establishing the optimal conditions for cell surv...

Page 55: ...bout the optical requirements of the chamber since the optical technology in use may range from a simple dissection scope to a multiphoton microscope In general however it is probably best to build a chamber with a glass microscope coverslip forming the bottom to ensure the best possible optical clarity Bath Electrode The simplest kind of bath electrode is a chlorided silver wire placed in the bat...

Page 56: ...ion of a computer in your electrophysiology rig is obligatory The minimum computer configuration requires a USB port for communicating with the MultiClamp 700B However in order to make full use of the power and convenience of your computer it is recommended that you also attach a digitizing interface such as the Digidata 1322A An interface allows you to generate command signals and save the data i...

Page 57: ...l configuration you can switch to I Clamp mode Advice on recording in I Clamp mode is given in the following section Sharp Microelectrode Recording Headstage and Holder Considerations Ensure the headstage is securely attached to the micromanipulator using one of the mounting plates on the headstage case Before attaching the pipette holder or inserting a pipette in the holder be sure to touch groun...

Page 58: ...pressing the syringe to 4 cc will give about 70 mbar of pressure This formula assumes about 2 m of 1 16 i d tubing is attached to the headstage holder Be aware that any air leaks in your system will nullify this estimate If you do not explicitly check for leaks the only indication that a leak exists may be an inability to get seals Some researchers prefer to apply pressure and suction by mouth In ...

Page 59: ...Drifting electrodes will cause a continual current drift off zero indicating that the electrodes probably need to be rechlorided Check the Seal Test checkbox and observe the Primary output Membrane Current on a scope the trace should resemble the top trace in Figure 4 1 Note the electrode resistance by checking the Resistance checkbox Lower resistances 2 4 MΩ are preferred for whole cell recording...

Page 60: ...ndicating that you have encountered a cell A slow increase probably means the tip is becoming clogged in which case you can try blowing it out with high pressure before advancing again at lower pressure 2 ms 250 pA IN BAT H PUS HE D AGAINS TCELL MOREPRE S SURE AGAINS TCELL GIGOHM S EAL Figure 4 1 Change in resistance while forming a seal When you are pushed up against a cell apply 50 100 mbar of s...

Page 61: ...ou should increase the value of the feedback resistor in the headstage in order to reduce instrument noise See Chapter 3 TUTORIAL 3 This is done under the Options button at the top of the MultiClamp 700B Commander After changing the feedback resistor you may need to readjust the Cp Fast and Cp Slow settings If you intend to apply voltage steps to the patch you may wish to use the Leak Subtraction ...

Page 62: ... 700B 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 Start with the Zap duration at 1 ms then depress the Zap button in the MultiClamp 700B Commander A successful break in will again look like that in Figure 4 2 If the patch is not disrupted the Zap duration can be increased and the Zap a...

Page 63: ... up with a reasonably square current step the amplitude of which reflects the input resistance of the cell See Chapter 3 TUTORIAL 4 The Whole Cell pF and MΩ values are estimates of respectively the cell s membrane capacitance and the access resistance due to the electrode plus any resistive contribution from the cell s contents The access resistance is typically about 3 times the electrode resista...

Page 64: ... perforated patch technique In this approach the very tip of the pipette is filled with a normal filling solution and the rest of the pipette is backfilled with the same filling solution to which 120 150 µg ml of the pore formers Nystatin Amphotericin B or Gramicidin from a stock solution of 30 mg ml in DMSO has been added Rae et al 1991 Yawo Chuhma 1993 Gramicidin has lower conductance than the o...

Page 65: ...nsients observed during amphotericin partitioning Low Noise Techniques The MultiClamp 700B 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 This means that any particular n...

Page 66: ...ly the thicker the wall is the lower the noise will be These expectations have been largely borne out by actual experiments Pipette glass can be obtained from specialty glass houses such as Clark Electromedical Instruments P O Box 8 Pangbourne Reading RG8 7HU England 073 573 888 Garner Glass 177 S Indian Hill Road Claremont CA 91711 USA 909 624 5071 Jencons Scientific Cherycourt Way Industrial Est...

Page 67: ...g up the outer wall of the pipette glass This is important since a thin film of solution on the outer surface of the glass produces a distributed resistance that interacts with the glass capacitance to produce a noise source that rises with frequency Since it becomes the dominant noise source it must be eliminated While many other hydrophobic substances have been used none to the best of our knowl...

Page 68: ...e applications it is recommended that an Axopatch 200B is used Electrode Holder The holders supplied with the MultiClamp 700B 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 Axon holders avoid metal and ...

Page 69: ...d be lifted until it is just under the surface of the bathing solution Signal Generator One last potential noise source to consider is the noise in the signal generator that provides the command In the MultiClamp 700B 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 sign...

Page 70: ...ttes is often unstable and can change erratically as the cell is penetrated In contrast the tip or junction potential of patch pipettes is stable and can be accurately measured and corrected for electronically There are some instances where micropipettes may be more useful If your study requires that the contents of the cell remain relatively intact second messenger systems for example then patch ...

Page 71: ...ass for recording Tip Resistance Tip resistance Re should be as low as possible and consistent with good impalements of the cell Low values of Re allow for greater stability and faster settling time of the microelectrode Stability Re of most microelectrodes changes with time and with current passing Re is affected not only by the magnitude of the current but also by its polarity In general microel...

Page 72: ...k of evaporation exposing the cells to the air unless a continuous flow of solution is provided across or through the preparation If evaporation is a problem try floating a layer of mineral oil on the surface of the solution If used this layer of oil will have the additional advantage of automatically coating the microelectrode as it is lowered into the solution Precautions must be taken to preven...

Page 73: ...eriment When the current is switched off the recorded potential should return to zero within a few milliseconds at most Some microelectrodes either return very slowly to zero potential or not at all These micropipettes should be discarded While the experiment is in progress occasionally check the resistance of the microelectrode Changes in tip potential are usually accompanied by changes in microe...

Page 74: ...rode into the cell then after 50 seconds of current at 1 nA or 1 s of current at 50 nA the change in concentration of A inside a cell 100 µm in diameter is 1 mM If A is an impermeant ion the cell may swell due to the inflow of water to balance the osmotic pressure The injection of a permeant ion such as chloride can significantly alter the equilibrium potential for that ion Impaling Cells Start wi...

Page 75: ...is point the cell can be impaled by pressing the Buzz button or the Clear Clear buttons If these fail vibrating the microelectrode tip by lightly tapping on the micromanipulator sometimes works When the electrode penetrates the cell there is a sudden change in the Membrane Potential trace reflecting the intracellular potential The voltage response to the Tuning steps will be slower and much larger...

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Page 77: ...f broad topics arranged in alphabetical order Because the MultiClamp 700B is effectively two instruments in one an Axopatch 1D and an Axoclamp 2B the topics are sometimes divided into two sections or refer to only voltage clamp or current clamp mode Please consult the Index if you are having trouble locating a particular item Note Before using this chapter it may be helpful to first read the entry...

Page 78: ... electrode One of two Audio Modes can be selected Direct Signal Monitoring The selected signal is relayed directly to the output speaker This mode is especially useful for monitoring spikes which are heard as audible clicks Voltage Controlled Oscillator VCO The voltage of the signal determines the frequency of a sine wave that is then directed to the output speaker This is useful if the signal of ...

Page 79: ...ections to the MultiClamp 700B 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 700B to use the computer s speaker Figure 5 1 Possible Audio configuration 1 Chapter 5 ...

Page 80: ...e front panel PHONES output or the rear panel AUDIO OUTPUT This allows dedicated use of the headphones or external speakers by the MultiClamp 700B Figure 5 2 Possible Audio configuration 2 MultiClamp 700B Theory and Operation Copyright 2005 Axon Instruments Molecular Devices ...

Page 81: ...same as option 2 except that now the MultiClamp 700B audio output is mixed with the computer s audio output to external speakers Figure 5 3 Possible Audio configuration 3 WARNING Never connect the computer s microphone jack to Audio connectors on the MultiClamp 700B This could lead to large voltages being sent to the MultiClamp 700B with the possibility of causing damage to its circuitry Chapter 5...

Page 82: ...significant voltage drop across the resistance of the grounding electrode The latter circumstance which normally arises only when voltage clamping large cells with large membrane currents is the situation for which the bath headstage is intended In a simple voltage clamp setup the voltage drop across the resistance of the bath grounding electrode Rb is indistinguishable from the membrane potential...

Page 83: ...he bridge as well as its contents i e Ringer s Solution versus 3 M KCl For a 1 cm long bridge 1 mm diameter 2 mm diameter Ringer s 10 2 kΩ 2 6 kΩ 3 M KCl 510 Ω 130 Ω Therefore to minimize Rb it would be best to eliminate the agar bridge and ground the preparation directly with a Ag AgCl pellet The pellet should be as large as practical and the area of it in contact with the solution should be maxi...

Page 84: ...strated in Figure 5 4 Figure 5 4 Bath headstage One electrode SENSE is a voltage sensing electrode It is placed in the bath near the cell surface It is connected to the virtual ground circuit by an agar bridge or similar of resistance Rb2 Since there is no current flowing through this electrode there is no voltage drop across Rb2 The other electrode IBATH with resistance Rb1 is also placed in the ...

Page 85: ...duct of I and the microelectrode resistance Re This unwanted IRe voltage drop adds to the recorded potential The Bridge Balance control can be used to balance out this voltage drop so that only the membrane potential is recorded The term Bridge refers to the original Wheatstone Bridge circuit used to balance the IR voltage drop and is retained by tradition even though operational amplifiers have r...

Page 86: ...ion the correction remains valid for any arbitrary waveform of injected current provided the microelectrode maintains a constant resistance Figure 5 5 Schematic bridge balance circuit and adjustment procedure Bridge Balance in the Bath Some investigators like to set Bridge Balance in the bath before attempting to impale cells This is to make it easier to see when a cell has been penetrated Check t...

Page 87: ...istinct The balancing procedure is the same as in the bath except that the trace appears more rounded because of the time constant of the cell membrane Because the Tuning pulse width is typically brief compared with the membrane time constant the voltage response looks like a series of ramping straight lines To make it easier to see the fast voltage step in Vp on an oscilloscope Figure 5 5B it is ...

Page 88: ...bound charges on the inside of the membrane The duration of the Buzz oscillation is set by the user 50 µs 500 ms The frequency of the oscillation is 10 kHz For some small cells a long duration Buzz can be deadly 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 Capacitan...

Page 89: ... to distortion of the signals of interest Injecting into the input of the headstage a current that directly charges the electrode capacitance bypassing the normal voltage clamp circuitry solves this problem Thus when the compensation is correctly adjusted the charge The MultiClamp 700B Commander provides two electrode compensation controls and discharge of the electrode capacitance is invisible to...

Page 90: ...actly equal Ip In this case Rf needs to supply no current and there is no transient on the output Whole Cell Capacitance Compensation When in whole cell mode a voltage clamp step must charge not only the electrode capacitance but also the capacitance of the cell Cm The decay time constant of the whole cell capacitance transient is determined by the product of Cm and the resistance in series Rs wit...

Page 91: ...djusting the SERIES RESISTANCE and WHOLE CELL CAP values in this circuit the current injected through C2 will supply the transient membrane current I These adjustments do not alter the time constant for charging the membrane Their function is to offload the burden of this task from the feedback resistor Rf In many cells even a small command voltage Vc of a few tens of millivolts can require such a...

Page 92: ...urden from Rf to C2 is illustrated in Figure 5 8 Figure 5 8 Using the injection capacitor to charge the membrane capacitance After perfect whole cell compensation is applied the current to charge the membrane capacitor is removed from the IRf trace and only the steady state current remains All of the transient current appears in the IC2 trace The IC2 trace in the figure was recorded using an oscil...

Page 93: ...negative default is 50 mV The Whole Cell Window Width is the duration of the window in multiples of Tau the fitted time constant of the whole cell transient over which the algorithm optimizes whole cell compensation The best setting depends on the cell type and is best found by trial and error As a general rule of thumb 1 x Tau works best for large cells with a highly distributed capacitance and 1...

Page 94: ...the effects of microelectrode capacitance in I Clamp mode This control is adjusted in the field in the I Clamp pane See also Bridge Balance Input Capacitance The capacitance Cin at the input of the headstage amplifier is due to the capacitance of the amplifier input itself Cin1 plus the capacitance to ground of the microelectrode and any connecting lead Cin2 Cin combined with the microelectrode re...

Page 95: ... value of Cin1 is thus reduced to well below its real value This eliminates the high frequency current loss through the power supply capacitance thereby increasing the bandwidth Since the power supply capacitance is present whether or not the power supply is bootstrapped there is no noise penalty due to implementing this technique Secondary Method for Neutralizing Cin In some cases the steps discu...

Page 96: ...ally prudent to reduce the Pipette Capacitance Neutralization setting slightly from the optimal in case the capacitance changes during the experiment Limitations of Capacitance Neutralization Use of capacitance neutralization is less desirable than physically minimizing Cin2 since the neutralizing circuit adds noise to the voltage signal This noise has been minimized in the CV 7 headstage of the M...

Page 97: ...tracellularly 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 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 Electrochemistry Using the Mu...

Page 98: ...er voltage clamp The potential at which dopamine and other catechol containing species such as epinephrine and norepinephrine oxidizes is approximately 0 15 V cf the silver silver chloride reference potential In order to accurately measure the voltammetric response of dopamine in solution the sensor is poised at a reducing potential between measurements and ramped to more oxidizing potentials to g...

Page 99: ...on Feedback Resistor Filter and Mode Although the MultiClamp 700B Commander provides some simple built in command stimuli e g via the Pulse button it is expected that most experiments will require more complex stimulus protocols These must be supplied by an external pulse generator or a computer program like pCLAMP External stimulus commands are supplied to the MultiClamp 700B via the COMMAND BNC ...

Page 100: ...nder Current Clamp Additivity of Commands All command stimuli applied by the MultiClamp 700B 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 Command Filter Frequency Prior to being applied to the cell the summed commands can be low pass filtered at a selectable frequency The Command Filter Freq...

Page 101: ...current that can be passed in I Clamp mode The value of this resistor is set in the Gains tab under the Options button See also External Command Inputs Headstage Noise Overload V Clamp Mode In V Clamp mode changing the value of the feedback resistor Rf in the headstage provides a method of changing the gain of the headstage Choice of the appropriate Rf involves a tradeoff between two competing fac...

Page 102: ...ntrinsic noise of the headstage circuitry Note Vcmd is limited to 10 V in the MultiClamp 700B which in turn limits the maximum amount of current that can be injected through the headstage resistor into the electrode For example with Rf 500 MΩ the maximum current that can be injected is 10 V 500 MΩ 20 nA These current limits are listed in the Options Gains panel of the MultiClamp 700B Commander Fig...

Page 103: ...should be in the range Rf 10 to Rf x 10 For example for a typical hippocampal pyramidal cell with an input resistance of 150 MΩ R Rf 50 MΩ is suitable Note that changing Rf in I Clamp mode changes the External Command Sensitivity for I Clamp Filters Low pass and high pass filters can be chosen to condition the Primary Output and Scope outputs The 3 dB frequency is selectable from a list in the Out...

Page 104: ...nd In the stop band the signal attenuates or rolls off with a characteristic steepness See Figure 5 10 noting the logarithmic frequency axis The steepness of the roll off at higher frequencies is determined both by the type of filter see below and the number of poles of the filter the larger the number of poles the faster the roll off The low pass on the Primary Output of the MultiClamp 700B are 4...

Page 105: ... the filter of choice when analyzing signals in the frequency domain e g when making power spectra for noise analysis The Butterworth filter has a sharp smooth roll off in the frequency domain but introduces an overshoot and ringing appearance to step signals in the time domain Choosing the Cutoff Frequency In practice there are two important considerations when selecting the filter cutoff frequen...

Page 106: ...e Tr in ms of a step input depends on fc in kHz approximately as Tr 0 35 fc This can be measured by applying Seal Test to the model BATH in V Clamp mode and looking at Primary Output Membrane Current while changing the filter setting Suppose you are interested in measuring action potentials for which you expect the 10 90 risetime to be about 0 4 ms You would then choose the filter cutoff frequency...

Page 107: ...mpensation See Chapter 5 EXTERNAL COMMAND INPUTS Grounding and Hum Methods for minimizing line frequency noise See also Noise Power Supply A perennial bane of electrophysiology is line frequency pickup often referred to as hum Hum can occur not only at the mains frequency but also at multiples of it In a well shielded enclosure the MultiClamp 700B has insignificant hum levels less than 0 01 pAp p ...

Page 108: ...in theory e g direct pickup earth loops in practice empiricism prevails Following the rules above is the best start The final hum level can often be kept to less than 0 1 pAp p One technique that should not be used to reduce hum is the delicate placement of cables so that a number of competing hum sources cancel out Such a procedure is too prone to accidental alteration Headstage Principles and pr...

Page 109: ... f PROBE Figure 5 11 Resistive feedback headstage The heart of the circuit is an operational amplifier op amp in the PROBE The behavior of this circuit depends upon two characteristics of an ideal op amp An op amp has infinite input resistance Therefore the current flowing out of the electrode Ie must equal the current If flowing through the feedback resistor Rf because no current is allowed to fl...

Page 110: ...the voltage clamp of the electrode must also be slow This is not the case for the following reason The PROBE op amp does everything it can to keep the voltage at its input equal to the command voltage at its input If the command is a rapid step then the voltage at the input i e at the back of the electrode is also a rapid step This means the voltage clamp of the electrode is fast The RC filtering ...

Page 111: ...K RESISTOR Current Clamp Circuit In I Clamp mode a separate headstage circuit is used called a voltage follower The essential features of a voltage follower are shown in Figure 5 12 A1 is an effectively infinite input resistance unity gain op amp the output of which is the pipette voltage Vp A2 is a summing amplifier used for injecting current into the cell The voltage across the headstage resisto...

Page 112: ... of the headstage The bath should not contact any other ground e g Signal Ground 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 Static Precautions The headstage can normally be s...

Page 113: ...he browser will open automatically if it is not already running and the relevant page will appear Holders Design use and maintenance of the HL U electrode holders supplied with the MultiClamp 700B The HL U series holder provides a universal fit for a very wide range of electrode diameters and will fit any of the U type headstages of Axon amplifiers Holder Design The barrel of the holder is made of...

Page 114: ...ured in place with a threaded collar Figure 5 13 Exploded view of the HL U holder The bore size of the HL U accepts pipettes with an outer diameter OD of 1 0 1 7 mm Pipettes are secured by a cone washer with an inner diameter ID that accommodates the pipette OD Color coding aids identification of the four sizes of cone washers 1 0 mm orange 1 3 mm clear 1 5 mm orange and 1 7 mm clear When the pipe...

Page 115: ...tional Ag AgCl Pellets The HL U holder will accommodate a 1 mm diameter Ag AgCl pellet that should provide many months of DC stable recordings The 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 sold as HLA 003 Figure 5 14 Ag AgCl pellet assembly Ho...

Page 116: ...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 reliably for several weeks Drifting or otherwise unstable offsets during experiments is suggestive of the need for rechloriding The chlorided region should be long enough so that the electrode solution does no...

Page 117: ...lar over the back of the barrel Adapters HLR U right angle adapters allow the HL U series holder to emerge at 90 from the headstage 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 Input Output Connections De...

Page 118: ...his special high gain output is useful for viewing very small extracellular signals External Command Potential VC or Current IC Auxiliary Potential if HS 2 headstage attached or Current if VG 2 headstage attached SCOPE The signal available here is the same as that at PRIMARY OUTPUT except that it can be independently low pass filtered using the Scope control in the Primary Output section of the ma...

Page 119: ... wish to mix the audio output of the MultiClamp 700B 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 AUDIO OUTPUT socket to the PC powered speakers AUDIO OUTPUT This output can be used in conjunction with AUDIO INPUT as described above It can also drive headphones or a remote powered speaker like the front panel Headpho...

Page 120: ...tting on top of a relatively large leak current Imagine for example a channel that opens during a 100 mV voltage step that is applied to a patch with a 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 700B cannot be turned up very far without saturating the amplifier but at a low gain setting the sing...

Page 121: ...n because it assumes that Rseal is constant for all voltage steps This may not be true if for instance the patch contains small channels or electrogenic transporters that do not produce discernible single channel events These will appear to be part of the seal current and may impart apparent non linear behavior to the seal For subtracting leak currents in whole cell recordings it is safer to use a...

Page 122: ...cell types and all recording configurations and have not encountered any problems with the transients causing damage of the cell membrane Model Cell PATCH 1U model cell is a standard accessory provided with the MultiClamp 700B 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 ...

Page 123: ... capacitance that increases the noise three to five times beyond what can be achieved with a good seal Instead of switches three separate plug positions have been provided and you can rotate the model cell into the position required With this technique the noise contribution of the model cell is still somewhat more than can be achieved with a good seal but the increase is less than 50 Figure 5 15 ...

Page 124: ...cuit designed for this purpose For white noise the rms noise is approximately one sixth the peak to peak noise The MultiClamp 700B Commander displays the rms noise on the Membrane Current signal in V Clamp mode after checking the Irms checkbox below the meters The measurement is made with a bandwidth of 30 Hz to 5 kHz 4 pole Butterworth filter See the table on page 92 in the Feedback Resistor sect...

Page 125: ...rove the bandwidth but increase the noise Electrode and Holder V Clamp Current noise increases markedly with electrode capacitance This can be minimized by coating the electrode and other strategies See Chapter 4 PATCH CLAMPING Increasing electrode resistance apparently decreases the current noise but this is due to the RC filtering effect of the electrode resistance in parallel with the electrode...

Page 126: ...ned above Electrode and Holder I Clamp Increasing Pipette Capacitance Neutralization increases the voltage noise for reasons similar to those just mentioned for Rs Compensation Although both of these compensation circuits increase the noise in the signal of interest they are most likely to be required in whole cell recordings where the dominant noise source is the cell In any case correction of Se...

Page 127: ...eckbox and manually adjusting the value to the left of the button See also Leak Subtraction Bridge Balance The purpose of this control is to zero the output that is to remove the DC voltage Output Zero works by sampling the current or voltage over a 70 ms time window immediately after pressing the button and then subtracting this value from all subsequent Primary Output signals Unlike Leak Subtrac...

Page 128: ...uration that occurs before the signal is conditioned By expanding transients Very fast saturating spikes e g uncompensated capacitance transients may be missed under visual inspection on an oscilloscope because they are too fast to be seen clearly The overload sensing circuitry in the MultiClamp 700B catches any signals that exceed saturation for longer than 10 µs and illuminates the OVERLOAD ligh...

Page 129: ...actured by Axon Instruments To prevent confusion Axon always uses current and voltage conventions based on the instrument s perspective 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 switch to the correct position Th...

Page 130: ...ge clamp is therefore Vm Vin Vout Vp 0 Vcmd Positive current flowing out of the electrode must then flow from the inside to the outside surface of the cell which means that it is outward current Polarity Summary for Different Recording Configurations Whole Cell Outside out Patch Positive current outward membrane current Membrane potential Vp Inside out Patch Positive current inward membrane curren...

Page 131: ...ame rating 4 Reconnect the power cord Glitches The MultiClamp 700B has been designed to minimize the effects of power supply 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 way to gain immunity from mains glitches is to eliminate them at the source Most...

Page 132: ...the features of the MultiClamp 700B Commander Note that telegraphs are active during Demo mode since they are communicated through software messaging MultiClamp Hardware This option only works when a functioning MultiClamp 700B is connected to a USB port on the computer that is running the MultiClamp 700B Commander The unique hardware Serial Number is identified by this operation Series Resistance...

Page 133: ...ial at the headstage The direction of the error will depend on the direction of current flow Worse the error will vary as the membrane current varies In extreme situations in the presence of voltage gated channels complete loss of control of membrane potential can occur 2 Dynamic voltage errors Following a step change in command potential the actual cell membrane potential will respond with an exp...

Page 134: ...box in the MultiClamp 700B Commander and pressing the Auto button to compensate the whole cell capacitance See Chapter 5 CAPACITANCE COMPENSATION The estimated Rs is the MΩ value displayed to the right of the manual adjust button under Whole Cell If Rs 10 MΩ and the maximum membrane current you anticipate is 100 pA the steady state voltage error will be at most 10 MΩ x 100 pA 1 mV which is probabl...

Page 135: ...ecause Rs compensation increases noise Adjusting Rs Compensation It is recommended that you practice adjusting Rs compensation with the PATCH 1U model cell before using compensation in a real experiment See Chapter 5 MODEL CELL Connect the CELL connector to the CV 7 headstage Set Primary Output to monitor Membrane Current Signal and increase output Gain to 10 Set the feedback resistor to 500 MΩ fo...

Page 136: ...er 5 CAPACITANCE COMPENSATION The estimated Rs which is the resistance we wish to compensate is the MΩ value displayed under the Whole Cell checkbox After compensation the trace will look like Figure 5 16 Figure 5 16 Uncompensated response with saturating transients Figure 5 17 After compensating transients MultiClamp 700B Theory and Operation Copyright 2005 Axon Instruments Molecular Devices ...

Page 137: ...eference Section 127 Figure 5 18 After setting Prediction 90 Correction 0 Figure 5 19 After setting Prediction 90 Correction 90 Figure 5 20 After optimizing Cf Rs and Cm to minimize transients Chapter 5 ...

Page 138: ...de of Vcmd This may only become noticeable after increasing the Primary Output Filter to 50 kHz bandwidth Reduce Prediction slightly if severe oscillations are observed Carefully increase the Correction value to equal that under Prediction A rather large transient should appear in the current at the beginning and end of the command step Its peak to peak amplitude should be 2 4 nA and it should und...

Page 139: ... kHz the Rs Compensation will be reduced substantially for conductance changes faster than 160 us Bottom line if you increase the Bandwidth value you can measure faster conductance changes but you sacrifice Rs compensation stability One tremendous advantage of the MultiClamp 700B is that you can choose to automatically disable or reduce Rs Compensation if oscillations should occur due to changes i...

Page 140: ...ection current that is injected through capacitor C2 Figure 5 20 Note that this C2 correction current does not improve the speed of clamping of the cell rather it charges the membrane capacitance as slowly as before but in a way that is invisible to the user because it bypasses the feedback resistor in the headstage Figure 5 21 Schematic whole cell compensation circuit MultiClamp 700B Theory and O...

Page 141: ...e pF and MΩ settings under Whole Cell and by the Prediction setting For example consider a whole cell voltage clamp situation where Rs 10 MΩ and Cm 50 pF and the resting membrane resistance Rm is very large with respect to Rs Assume that Whole Cell pF and MΩ are set at 10 MΩ and 50 pF respectively so that the whole cell capacity transient is perfectly canceled If the Prediction control is 0 the si...

Page 142: ...will now respond to a step voltage command with a 10 90 risetime of about 22 µs and will settle to within 1 of its final value in less than 50 µs Saturation Effects Note that the equation presented above for Vcp i e the command potential plus Prediction signal can be used to define the maximum allowable Prediction for a given size step voltage command This limit should not be confused with limitat...

Page 143: ... headstage capacitor C2 is modified appropriately so that it will continue to cancel the whole cell capacity transient despite the fact that the speed of this transient has increased This is simply accomplished by reducing the time constant of this signal as Prediction is increased If the circuitry worked perfectly and if the whole cell capacity transient had been perfectly canceled with 0 Predict...

Page 144: ...in using Correction with the Bandwidth set at 10 kHz or less However once the desired level of Correction has been achieved it is usually possible if desired to significantly increase the Bandwidth setting 30 kHz is usually quite achievable for 90 Correction Continuing with the example considered above Rs 10 MΩ Cm 50 pF a 90 Correction setting will reduce voltage errors in the true membrane potent...

Page 145: ...e Correction controls set at different values For example for a 200 mV step command Prediction should be limited to about 80 to avoid saturation See Saturation Effects above However it is usually possible to compensate series resistance up to 90 to 95 or more by use of the Correction control In other patch clamps the issue of saturation would limit the amount of compensation used for ionic current...

Page 146: ...ction of the Bandwidth control Limitations of Rs Compensation Series resistance compensation is an attempt to electronically reduce the effect of the electrode resistance Because of practical limitations it is never perfect Even if 100 compensation could be used with stability this would only apply to DC and medium speed currents Very fast currents cannot be fully corrected For best results the ce...

Page 147: ...of mouse gliders and clicks in the MultiClamp 700B Commander software The SoftPanel is merely a hardware extension of the Commander and replicates the many Commander control functions of the MultiClamp 700B The SoftPanel comes with a magnetic overlay with pre defined functions assigned to the various knobs and buttons However the SoftPanel can easily be re configured in the MultiClamp 700B Command...

Page 148: ... by pressing the button in the V Clamp pane The conventional method for rupturing a membrane patch to go to whole cell recording is to apply a pulse of suction Sometimes this method damages the cell Zap provides an alternative method It applies a large 1 V voltage pulse that ruptures the patch presumably by causing dielectric breakdown of the membrane The Zap duration can be varied it is best to u...

Page 149: ...700B Commander to recheck the hardware configuration press the Select Device button in the toolbar See Chapter 5 SELECT DEVICE Some common problems are listed below Symptom The MultiClamp 700B is not responding to commands The Status light is not flashing Possible causes The USB cable is not plugged in properly or is defective The PC s USB port is defective Select Device has been set to Demo rathe...

Page 150: ...e outside of the chamber is dry Set Pipette Capacitance Neutralization to zero Symptom Extraneous noise is present in the Primary Output signal Pipette Offset is drifting rapidly Possible cause The Ag AgCl pellet or Ag wire in the electrode holder may be defective Dirt or corrosion may have built up in the holder or headstage connector socket Suggestions Check the DC stability of the pellet and re...

Page 151: ...uency 50 60 Hz Fuse 5 mm x 20 mm 2A 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 7 Headstage Dimensions 4 06 x 8 38 x 2 03 cm 1 6 x 3 3 x 0 8 Voltage Clamp Gain Rf 50 GΩ 5 GΩ 500 MΩ 50 MΩ 10 kHz Noise 8 pole Bessel filter 50 G 0 28 pArms 5 G 0 9 pArms 500 M 1 4 pArms 50 M 3 0 pArms Specifications ...

Page 152: ...nd 200 4000 µs Whole cell capacitance compensation Cm from 1 pF to 100 pF and Rs from 400 k to 1000 M on 500 M range Cm from 2 5 pF to 1000 pF and Rs from 100 k to 100 M on 50 M range Series Resistance compensation Bandwidth is adjustable from 0 32 to 16 kHz Series resistances corrected varies from 0 4 to 1000 M on 500 M range and 0 1 to 100 M on 50 M range Current Clamp Rise time 10 µs for load o...

Page 153: ...Tune frequency is selectable from 2 to 1000 Hz Pulse duration is selectable from 0 1 to 500 ms Buzz amplitude is fixed at 15 V signal to the headstage capacitor but with selectable 0 05 to 500 ms duration Clear amplitude is fixed at 15 V signal to the headstage capacitor DC Holding Commands Voltage Clamp 1000 mV range in 30 µV steps Auto Pipette Offset adjusts DC holding potential to zero Membrane...

Page 154: ...3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 60 90 120 150 180 210 240 270 300 330 360 390 420 450 600 900 1k2 1k5 1k8 2k1 2k4 2k7 3k 3k3 3k6 3k9 4k2 4k5 6k 9k 12k 15k 18k 21k 24k 27k 30k 33k 36k 39k 42k 45k Bypass Highpass single pole Bessel frequencies Hz DC 0 1 1 3 10 30 100 300 Secondary Output Filters Lowpass single pole Bessel filter fixed at 10 kHz frequency or Bypass Scope Filter Lowpass two ...

Page 155: ...elay to return from V Clamp 20ms 500 seconds in 10 ms steps this can also be done manually or with External Mode BNC Switching Speeds Auto from I Clamp to V Clamp 0 5 ms Auto from V Clamp to I Clamp 22 ms Mode switching performed manually with the mouse keyboard or SoftPanel interface will always be slower than automatic switching due to delays in computer operating system communication Add approx...

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Page 157: ...h a single microelectrode In Voltage and Patch Clamping with Microelectrodes Smith T G Lecar H Redman S J Gage P W Eds Williams Wilkins Baltimore 1985 Furman R E and Tanaka J C Patch electrode glass composition affects ion channel currents Biophys J 53 287 292 1988 Hamill O P Marty A Sakmann B and Sigworth F J Improved patch clamp techniques for high resolution current recording from cells and cel...

Page 158: ...dition Plenum Press New York 1995 Sherman Gold R The Axon Guide for Electrophysiology Biophysics Laboratory Techniques Axon Instruments Foster City CA 1993 Available at http www axon com Yawo H and Chuhma N An improved method for perforated patch recordings using nystatin fluorescein mixture Jap J Physiol 43 267 273 1993 MultiClamp 700B Theory and Operation Copyright 2005 Axon Instruments Molecula...

Page 159: ...p to resolve a problem there are several ways to contact Axon Instruments Molecular Devices World Wide Web www axon com Phone 1 800 635 5577 Fax 1 510 675 6300 E mail axontech axon com Questions See Axon s Knowledge Base http support axon com Technical Assistance ...

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Page 161: ...pping of the product to the customer If the shipment is to a location outside the United States the customer will be responsible for paying all duties taxes and freight clearance charges if applicable 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...

Page 162: ...0B is a solidly built instrument designed to survive shipping around the world However in order to avoid damage during shipping the MultiClamp 700B must be properly packaged In general the best way to package the MultiClamp 700B is in the original factory carton If this is no longer available we recommend that you carefully wrap the MultiClamp 700B in at least three inches 75 mm of foam or bubble ...

Page 163: ...e to avoid but it is inexpensive compared to the cost of repairing an instrument that has sustained shipping damage It is your responsibility to package the instrument properly before shipping If the packaging is inadequate and the instrument is damaged during shipping the shipper will not honor your claim for compensation Warranty and Repair Service ...

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Page 165: ... will not be accepted Name of registered owner ________________________________________________________________________ Department ___________________________________________________________________________________ University Institute _____________________________________________________________________________ Street address _______________________________________________________________________...

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Page 167: ...03 Application of Council Directives EC EMC Directive 89 336 EEC as amended EC Low Voltage Directive 73 23 EEC as amended Harmonized Standards to which Conformity is Declared EMC EN 61326 1 1997 A1 1998 A2 2001 EN 55011 CISPR11 1998 AS NZS 2064 1997 Safety EN 61010 1 2001 I the undersigned hereby declare that the equipment specified above conforms to the above Directives and Standards Authorized S...

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Page 169: ...detrimental to the environment Safe Environmental Conditions 1 Indoor use 2 Mains supply fluctuations not to exceed 10 of the nominal voltage 3 Temperature between 5 ºC and 40 ºC 4 Altitude up to 2000 m 5 This instrument is designed to be used under laboratory conditions Operate in a clean dry environment only Do not operate in a wet or damp environment Static Precautions If you are in a laborator...

Page 170: ...ent pas dépasser 10 de la tension nominale 3 Température entre 5 C et 40 C 4 Altitude jusqu à 2000 m 5 Cet appareil a été étudié pour l emploi en laboratoire et il doit être situé dans un environnement sec et propre Ne pas l utiliser dans un environnement mouillé ou humide Précautions statiques Le headstage peut être maniée sans danger Cependant dans un laboratoire avec un niveau élevé d electrici...

Page 171: ...ere Betriebsbedingungen 1 Verwendung in Innenräumen 2 Netzschwankungen darf nicht 10 der Nennspannung überschreiten 3 Temperatur zwischen 5 C und 40 C 4 Höhe bis zu 2000 m 5 Dieses Instrument ist für den Gebrauch unter Laborbedingungen vorgesehen Nur in sauberer trockener Umgebung in Betrieb setzen Nicht in nasser oder feuchter Umgebung in Betrieb setzen Schutzmaßnahmen gegen statische Aufladung D...

Page 172: ...tro no deben exceder 10 del voltaje nominal 3 Temperatura entre 5 C y 40 C 4 Altitud hasta 2 000 m 5 Este instrumento está diseñado para ser usado en condiciones de laboratorio Debe operarse únicamente en un ambiente limpio y seco No lo use en un ambiente húmedo ni mojado Precauciones contra la estática El headstage puede manejarse con seguridad bajo condiciones normales Sinembargo si usted se enc...

Page 173: ...1 93 98 101 102 111 114 115 122 141 Holders 58 103 Clear 65 78 87 143 Correction 30 127 128 129 134 135 Current Clamp Capacitance Compensation 78 79 80 83 89 90 110 118 122 124 126 Model Cell 112 125 Whole cell 51 81 Electrochemistry 87 External Command 78 84 89 90 91 93 97 107 External Command Inputs 78 84 89 91 93 97 107 Filter 89 90 95 97 128 142 144 Functional Checkout 6 Gain 23 24 110 118 141...

Page 174: ...1 Voltage Selection 121 159 Practical Guide Forming a Gigaseal 49 Interfacing a Computer 46 Low Noise Techniques 48 55 Perforated patch Recording 54 Sharp Microelectrode Recording 47 59 85 86 115 Whole cell Voltage Clamp Recording 51 Prediction 30 127 128 129 131 132 133 134 135 Primary Output 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33 37 49 64 65 76 86 93 96 97 110 117 128 140 144 Pulse 30 3...