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Guide to Electrophysiological Recording

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39

Chapter 4

Once the gigohm seal is established, the rectangular current pulse will disappear
entirely and be replaced by capacitance transients in synchrony with the rising and
falling edges of the command pulse (Figure 3.1, lowest trace). These can be canceled
by pressing the “Cp Fast: Auto button”. You may need to manually adjust the
capacitance (pF) and time constant (µs) parameters for optimal cancellation. (See
Chapter 3,

TUTORIAL 3

.) A slower component of the transients may be reduced using

the Cp Slow controls.

If you wish to remain in cell-attached mode (for example, to record single-channel
currents) you 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 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 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. The
operation of this feature is described in Chapter 3,

TUTORIAL 4

.

Whole-cell Voltage Clamp Recording

Obtain a gigaseal as described above. The electrode should contain a low Ca

2+

solution (

i.e.

, buffered with EGTA to ~ 100 nM) that mimics the intracellular milieu,

and the electrode resistance should be low (~3-4 M

Ω

). During or immediately after

seal formation, set the holding potential (Holding:) in the MultiClamp Commander to
the anticipated resting potential of the cell (typically ~ –60 or –70 mV). Alternatively,
the holding potential can be set in Clampex.

A pulse of strong suction is applied to rupture the cell membrane. This can again be
done by mouth suction or by a syringe. Mouth suction tends to give the best control.
Apply brief (~0.5 s) pulses of suction, starting gently (

e.g.

~80 mbar) and increasing

the suction after every couple of pulses until a large capacitance transient suddenly
appears (Figure 3.2). If you are using a 10 cc syringe, draw back on the plunger until

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Содержание MultiClamp 700A

Страница 1: ...yright 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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Страница 3: ...OROUGHLY CALIBRATED 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 ...

Страница 4: ...umber MultiClamp 700A Year of Manufacture 2000 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 55011 1991 CISPR11 1992 Amd 1 2 1996 AS NZS 2064 1997 EN 50082 1 1997 Safety EN 61010 1 2001 I the undersigned hereby declare that the equipment specified above conforms to t...

Страница 5: ...he 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 The headstage can normally be safely handled However if you are in a laboratory where static is high i e you hear and feel crackles when you touch t...

Страница 6: ... 1 Emploi à l intérieur 2 Fluctuations du réseaux d alimentation ne doivent 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 sa...

Страница 7: ... Umweltsichere 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 ...

Страница 8: ...ior 2 Fluctuaciones eléctricas en la fuente de suministro 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 segurida...

Страница 9: ...6 Setting Parameters in the MultiClamp Commander 6 Toolbar Buttons in the MultiClamp Commander 8 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 Giga Seal Configuration 18 Tutorial 4 Whole Cell Conf...

Страница 10: ...e cell Voltage Clamp Recording 39 Perforated patch Recording 42 Low Noise Techniques 43 Sharp Microelectrode Recording 47 Sharp Microelectrode or Patch Electrode 48 Microelectrode Properties 48 Filling Solutions 52 Impaling Cells 52 CHAPTER 5 REFERENCE SECTION 55 Audio Monitor 56 Bath Headstage and Electrodes 60 Bridge Balance 63 Buzz 66 Capacitance Compensation 67 Capacitance Neutralization 73 Cl...

Страница 11: ...cilloscope 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 REFERENCES 141 WARRANTY AND REPAIR SERVICE 143 SHIPPING 145 INDEX 149 ...

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Страница 13: ...llows the user to remotely switch between low noise patch clamp recording using the Axopatch like headstage circuit and true fast current clamp recording using the Axoclamp like headstage circuit With two headstages the MultiClamp 700A effectively provides four amplifiers in one unit The MultiClamp 700A is essentially an analog in analog out instrument like conventional amplifiers by Axon Instrume...

Страница 14: ...control of an amplifier is an unusual step forward for some users If computer mouse control is unsettling consider the optional SoftPanel device to control the MultiClamp 700A The SoftPanel is essentially a hardware extension of the MultiClamp Commander software Knobs and buttons replace the mouse glider and clicks For more information visit our website or call Axon Technical Support The MultiClam...

Страница 15: ... 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 700A main unit 2 Two CV 7A headstages the MultiClamp 700A with one headstage is optional 3 Serial control cable RS232 null modem and DB9 to DB25 adapter if necessary 4 MultiClamp Commander host software from CD or...

Страница 16: ...apter if the computer port is a 9 pin type 2 Connect the CV 7A headstages to HEADSTAGE 1 and HEADSTAGE 2 rear panel connectors 3 Power up the MultiClamp 700A The front panel POWER light light emitting diode or LED should illuminate as well as either the VOLTAGE CLAMP or CURRENT CLAMP LED for each channel Installing the MultiClamp Commander 1 Run the MultiClamp Commander installer This will install...

Страница 17: ...the back of the MultiClamp 700A default setting is 1 5 If the program is unable to find a valid Device Number try the following Select a different Serial Port and try scanning again Check that the MultiClamp 700A is switched on and that the serial cable is connected properly 6 Press the OK button the main MultiClamp Commander window should appear If installed correctly the COM port and Device numb...

Страница 18: ...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 2 Toggle the Channel 1 Mode and Channel 2 Mode buttons switching repeatedly between Voltage Clamp VC and Current Clamp I 0 IC modes Figure 1 3 The tabs immediately below the mode switches will change ...

Страница 19: ... 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 horizontally changes the time constant parameter τs Simultaneously pressing the Shift or Ctrl key magnifies the effect 10 fold and 100 fold respectively 2 Entering text directly Position the ...

Страница 20: ...play a Tool Tip for the button Most 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 Commander The Quick Select buttons can be assigned to a particular set of parameter settings to facilitate rapid loading This might be useful for experiment...

Страница 21: ...e 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 2 To reduce extraneous noise the CV 7A must be shielded This can be accomplished using aluminum foil which should be loosely but completely wrapped around the headstage Most importantly the input of the CV 7A should not make contact with the foil A great al...

Страница 22: ...teps 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 1 Connect an oscilloscope to the front panel SCALED OUTPUT or SCOPE OUTPUT BNC 2 Synchronize the oscilloscope by connecting to the rear panel SYNC OUTPUT BNC Press the Reset to Program Defaults button on the MultiCl...

Страница 23: ... amplitude following the transient This should be 500 mVp p 50 mV 5 G Range 1 Change the feedback resistor to 5 G 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 Defaults button By default the 500 M range is selected 2 Check Seal Test and set the amplitude to 25 mV 3 Plug the CELL connector of the PATCH 1U model cell into the CV 7A headstag...

Страница 24: ...l connect you to the On line Help which describes many of the functions of the MultiClamp 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 Commander because this information is better provided in an interactive way using the On line Help Rather the purpose of this manual is to provi...

Страница 25: ...ugh this chapter is primarily directed at inexperienced electrophysiologists it may also be useful for experienced researchers who desire a simple introduction to the features of the MultiClamp 700A It is recommended that the tutorials are done in order because later tutorials assume knowledge gained in earlier ones Check List These tutorials require the following 1 MultiClamp 700A main unit plus ...

Страница 26: ...tors 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 electrode resistor to ground PATCH 10 GΩ patch resistor to ground Approximately 5 pF stray capacitance to ground CELL 10 MΩ electr...

Страница 27: ...iClamp 700A to the oscilloscope The oscilloscope display should initially be set at 0 5 V division and 2 ms division Triggering should be set to Line Alternatively connect a BNC cable from the Channel 1 Scaled Output to and Analog Input on the front panel of a Digidata digitizer for monitoring on the Scope Window of Clampex 4 Press the Pipette Offset button while looking at the oscilloscope Figure...

Страница 28: ...der Scaled Output in the Output Signals section Figure 2 5 Therefore the resistance of the model electrode is calculated from Ohm s Law to be R V I 10 mV 1 nA 10 MΩ Alternatively check the Resistance checkbox under the Channel 1 meters Figure 2 6 The resistance is displayed on the meter Uncheck the box when done DC fluctuations in the signal are due to pulses from the MultiClamp Commander for calc...

Страница 29: ...utorial 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 Figure 2 8 Note that the Scaled Output signal displayed on the oscilloscope is now Membrane Potential 1 V V as shown in the Output Signals section of the MultiClamp Commander 3 Press the Pipette Offset button T...

Страница 30: ...onfiguration 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 This connects a 10 GΩ resistor to ground simulating a gigaseal 2 One of the advantages of a gigaseal is that the recording noise is dramatically reduced enabling single channel measurements...

Страница 31: ...te the information provided under Experiment Type and Range in the Gains panel Figure 2 11 Select 50 GΩ feedback resistor and then close this panel 4 Note that the noise trace on the oscilloscope is now about 150 mVp p However the Scaled Output gain shown under Output Signals is now 50 V nA so the noise is 3 pAp p a 3 fold reduction compared with before This is still quite noisy for recording sing...

Страница 32: ...t in the output although in practice it is best to avoid doing this on the principle that all aspects of a biological signal are potentially interesting 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...

Страница 33: ...ws See the figure below While holding down the Shift key to magnify the movement see Chapter 2 use the glider sliding the mouse horizontally and vertically to change the values of the time constant and capacitance respectively Alternatively you can place the mouse cursor over each parameter display in turn and use the glider to adjust each individually Figure 2 14 Notice that you can change the am...

Страница 34: ...g on the model cell has only a very minor slower transient 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 Clear steps should now be visible on the oscilloscope wit...

Страница 35: ... more easily seen if the oscilloscope is triggered using the SYNC output of the MultiClamp 700A Figure 2 16 The fast component of the transients reflects the simulated electrode capacitance 5 pF while the slow component reflects the 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 re...

Страница 36: ...and series resistance MΩ parameters See Figure 2 18 below It will be easier to do this while holding down the Shift key to magnify the effect of mouse movement Figure 2 18 It should be possible to compensate completely the slow transient The optimal values will be around 30 pF the model cell capacitance and 10 MΩ the model electrode resistance Note that a small fast transient may reappear after th...

Страница 37: ...btracting a constant DC offset without regard to the voltage command To illustrate its use switch off Leak Subtraction and check and press Output Zero with Holding set to a large negative value as described in step 3 of this Tutorial The Scaled Output trace is baselined but unlike with Leak Subtraction the step due to Seal Test is not subtracted 7 The series resistance Rs which typically originate...

Страница 38: ...sed beyond about 80 the transients become larger and then rapidly escalate into a full blown oscillation The art of Rs compensation is to choose a combination of Bandwidth Prediction and Correction that provides maximal compensation without oscillation Full details are given in SERIES RESISTANCE COMPENSATION in Chapter 5 9 The MultiClamp 700A is designed to be used with an external pulse generator...

Страница 39: ...A unit illuminates and Scaled Output displays Membrane Potential 3 Check the open box next to Holding and using glider control vary the holding current nA while looking at Scaled Output on the oscilloscope or the V mV meter The model membrane potential varies smoothly with Holding current Figure 2 22 4 Switch off Holding and check the Tuning checkbox while monitoring Scaled Output on the oscillosc...

Страница 40: ...he case of whole cell voltage clamp Tutorial 4 electrode series resistance can introduce errors to current clamp recordings and needs to be compensated electronically In current clamp mode Rs is compensated using Bridge Balance Figure 2 25 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...

Страница 41: ...ette Capacitance Neutralization pF value while monitoring Scaled Output on the oscilloscope Figure 2 26 Note that as you increase the value beyond about 3 pF damped oscillations start to appear at the beginning of each sawtooth Figure 2 27 If you go further full blown oscillations develop As in the case of Rs compensation in voltage clamp mode the art of pipette capacitance neutralization is to in...

Страница 42: ...ion ms while monitoring the effect on Scaled Output Figure 2 28 8 Switch on both Holding and Tuning Observe Scaled Output on the oscilloscope while pressing the I 0 button Figure 2 29 I 0 is a special mode of current clamp in which all command inputs are disconnected With the model cell the Scaled Output Membrane Potential returns to near 0 mV when I 0 is pressed In a real cell the Membrane Potent...

Страница 43: ...chnical details for reference 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 circumst...

Страница 44: ...ns and or O2 to maintain the metabolic viability of the cells Some cells e g those in retinal slices 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 y...

Страница 45: ...ics Again it is difficult to generalize about 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 ...

Страница 46: ...acing a Computer Because the MultiClamp 700A is a computer controlled instrument the installation of a computer in your electrophysiology rig is obligatory The minimum computer configuration requires a serial port for communicating with the MultiClamp 700A 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 s...

Страница 47: ... whole cell 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 t...

Страница 48: ... 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 this case it might be useful to roughly calibrate yo...

Страница 49: ...cell recording to minimize series resistance but if the resistance is too low it can be difficult to obtain a gigaseal Higher resistances 5 MΩ are obviously necessary for sealing onto smaller cells or processes Apart from these basic rules choice of the appropriate electrode resistance is largely a matter of experience and experimental design The method of approaching the cell depends upon whether...

Страница 50: ...forming a seal When you are pushed up against a cell apply 50 100 mbar of suction negative pressure to the pipette holder At the same time steadily increase the holding potential towards 60 or 70 mV doing this usually helps seal formation There should be a rapid increase in the resistance Release the suction when the resistance reaches a gigohm The resistance often continues to increase slowly ove...

Страница 51: ...Clamp 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 The operation of this feature is described in Chapter 3 TUTORIAL 4 Whole cell Voltage Clamp Recordin...

Страница 52: ...reak in will again look like that in Figure 3 2 If the patch is not disrupted the Zap duration can be increased and the Zap applied a second time and so on Some investigators have found that the application of moderate suction while the Zap pulse is given results in a higher incidence of successful patch disruption The reappearance of the original rectangular pulse either means that you have lost ...

Страница 53: ...suction or more dangerously by brief pulses of pressure Whenever voltage clamping in whole cell mode it is advisable to use Rs compensation to minimize the voltage drop across the access resistance A common mistake is to assume that this Rs error is small so as to avoid the fiddly process of setting Rs compensation This is false economy Rs errors can be surprisingly large and can easily render you...

Страница 54: ...tions Ebihara et al 1995 A cell attached seal is then formed on the cell Over a 5 30 minute time period myriad tiny cation selective voltage independent channels are inserted in the membrane patch These channels allow small ions to equilibrate between the cell and the pipette allowing the cell to be voltage clamped through the open channels Since substances as large as or larger than glucose will ...

Страница 55: ...of the sum of the individual squared rms noise sources This means that any particular noise source that is large will dominate the total noise and make other noise sources insignificant Therefore all potentially contributing noise sources must be minimized Specifically the headstage the pipette glass the holder and the seal contribute significantly even under circumstances where extraneous noise p...

Страница 56: ...medical 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 Estate Stanbridge Road Leighton Buzzard LU7 8UA UK 0525 372 010 Sutter Instrument Company 51 Digital Drive Novato CA 94949 USA 415 883 0128 Each type of glass has unique advantages and disadvantages Alumino...

Страница 57: ... 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 knowledge produces as low noise as does Sylgard 184 Sylgard also decreases the capacitance of the pipette wall and...

Страница 58: ...stage of the MultiClamp 700A because of technical limitations with the digital circuitry 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 be...

Страница 59: ...cts with the pipette capacitance to produce a noise source that rises with frequency In order to minimize noise when recording from excised membrane patches the electrode tip should 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 700A we...

Страница 60: ...ance a patch pipette used for voltage recording will have a better frequency response and lower noise level than a micropipette Furthermore the tip potential of high resistance intracellular micropipettes 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 elect...

Страница 61: ...ting glass 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 ...

Страница 62: ...ss loss of performance When working with very low solution levels there is a risk 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 micro...

Страница 63: ...the experiment 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...

Страница 64: ...l For example if current is passed by the flow of ion species A from the microelectrode 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...

Страница 65: ...l At this 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...

Страница 66: ......

Страница 67: ...umber of broad topics arranged in alphabetical order Because the MultiClamp 700A 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 th...

Страница 68: ... cell since it obviates the need to look at an oscilloscope while manipulating the 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...

Страница 69: ...aking 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 Figure 4 1 Possible Audio configuration 1 ...

Страница 70: ...000 2001 Axon Instruments Inc 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 ...

Страница 71: ...is 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 Figure 4 3 Possible Audio configuration 3 WARNING Never connect the computer s microphone jack to Audio connectors on the MultiClamp 700A This could lead to large voltages being sent to the MultiClamp 700A with the possibility of causing damage to its circuitry...

Страница 72: ...p 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 That is the potential ...

Страница 73: ...ontact with the solution should be maximized With this kind of bath electrode you should avoid perfusing the bath with solutions containing different chloride activities The DC offset of an Ag AgCl pellet changes with chloride activity In order to minimize Rb when using an agar bridge it is best to fill the bridge with 3 M KCl instead of Ringer s solution When the agar bridge is filled with 3 M KC...

Страница 74: ... 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 bath This electrode carries the ionic current The feedback action of the operational amplifier ensures that the potential at the SENSE electrode is equal to the potential at the posit...

Страница 75: ...ance the IR voltage drop and is retained by tradition even though operational amplifiers have replaced the original circuitry The technique is illustrated schematically in Figure 4 5A A differential amplifier is used to subtract a scaled fraction of the current I from the voltage recorded at the back of the microelectrode Vp The scaling factor is the microelectrode resistance Re The result of this...

Страница 76: ...led Output Press the Auto Bridge Balance button the fast voltage steps seen at the start and finish of the current step should be eliminated You may need to manually adjust the Bridge Balance MΩ value for optimum balance The MΩ value is the resistance of the electrode Bridge Balance in the Cell The Bridge Balance should be frequently checked when inside a cell because the electrode resistance can ...

Страница 77: ...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 4 5B it is recommended that the scope input be AC coupled to remove the resting membrane potential from the signal The scope gain can then be turned up without the annoying offset The MΩ value found by Bridge Balance is the resistance of the electrode which may be s...

Страница 78: ... tips When used while the tip of the microelectrode is pressing against the membrane Buzz may also cause the micropipette to penetrate the cell The exact mechanism is unknown but it may involve attraction between the charge at the tip of the electrode and 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 oscillatio...

Страница 79: ...ltage clamp step is applied to an electrode the clamp must provide a spike of current at the start and finish of the step to charge and discharge the capacitance of the electrode Cp The main problem with these spikes is that they may saturate the headstage circuit or later circuits leading to distortion of the signals of interest Injecting into the input of the headstage a current that directly ch...

Страница 80: ...de capacitance that can be represented by a lumped capacitance at the headstage input This is the major part of Cp A small amount of Cp can only be represented as a capacitor with a series resistance component This takes longer to charge to its final value and is compensated by the Cp Slow controls C1 1 pF Rf R p OPEN CIRCUIT 10 Vp FAST MAG SLOW MAG SLOW FAST I p Cp Vp I C1 I τ τ Figure 4 6 Pipett...

Страница 81: ...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 with Cm If Rs and Cm are both reasonably large the resultant capacitance transient can last for several milliseconds perhaps distorting the rising phase of biologically interesting currents Furthermore as in the case of the electrode...

Страница 82: ...tor Rf In many cells even a small command voltage Vc of a few tens of millivolts can require such a large current to charge the membrane that it cannot be supplied by Rf The headstage output saturates for a few hundred microseconds or a few milliseconds thus extending the total time necessary to charge the membrane This saturation problem is eliminated by appropriate adjustment of whole cell capac...

Страница 83: ...tial outputs on the MultiClamp 700A would look like the IRf and Vc 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 Use of an independent electrode in the cell would show that the cell charging rate is not affected by these adjustments The pF and MΩ values found by the MultiClamp Commander for...

Страница 84: ...tance Compensation Although the algorithm used by the Auto button is reasonably robust and is likely to work under most circumstances it may sometimes be necessary to manually adjust the Cp Fast Slow or Whole Cell compensation This is done by using the dual controls or by entering values directly It is recommended that you practice using these controls with the PATCH 1U model cell The best strateg...

Страница 85: ...microelectrodes with the lowest possible resistance compatible with stable recording and take steps to minimize the contribution to Cin by the capacitance of the microelectrode In practice this means using patch electrodes where possible or using sharp microelectrodes with minimal capacitance See Chapter 4 SHARP MICROELECTRODE RECORDING Electronically neutralize Cin The second approach has been im...

Страница 86: ...ce compensation negative capacitance or capacitance neutralization A compensation amplifier at the output of the unity gain buffer drives a current injection capacitor connected to the input At the ideal setting of the compensation amplifier gain the current injected by the injection capacitor is exactly equal to the current that passes through Cin2 to ground Adjusting Capacitance Neutralization C...

Страница 87: ...een minimized in the CV 7A headstage of the MultiClamp 700A by using low noise amplifiers and small injection capacitors but it is still significant It is important to recognize that the capacitance neutralization circuit is not more than 90 effective even for ideal microelectrodes This is because of the finite frequency responses of the headstage amplifiers and the capacitance neutralization circ...

Страница 88: ...or two purposes Clearing blocked microelectrodes If the microelectrode resistance in the bath seems much higher than it should be the electrode can often be cleared by rapidly toggling the Clear switch from plus to minus Because of the large current passed this should only be done extracellularly Penetrating cells Sometimes microelectrode tips press against the cell membrane but fail to penetrate ...

Страница 89: ...is relatively specific for different chemical classes of compounds In short the technique generates a chemical fingerprint for each compound of interest Furthermore the current that is derived from the oxidation or reduction of these compounds is directly proportional to the concentration Two methods are used for making electrochemical measurements cyclic voltammetry and amperometry Cyclic voltamm...

Страница 90: ... used for measuring quantal release of electro active chemicals from vesicles 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 volt...

Страница 91: ...nt 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 External Command Sensitivity External Command Sensitivity is a scaling parameter that is set in the Gains tab under the Options button In V Clamp mode the purpose of External Command Sensitivity is to scale down the...

Страница 92: ...he summed commands can be low pass filtered at a selectable frequency The Command Filter Frequency is set in the General tab under the Options button The selectable frequency is the 3 dB cutoff frequency of a 4 pole Bessel filter Two filter settings are provided for each Channel one for V Clamp the other for I Clamp This feature is provided because sometimes is desirable to round off the commands ...

Страница 93: ...rger values of Rf are more suited to patch recordings where the noise is more critical and the currents are smaller whereas smaller values of Rf are more suited to whole cell recordings with their larger currents The following table summarizes these properties for different values of Rf Feedback Resistor Experiment Type Range Noise 50 MΩ Whole Cell 1 200 nA 3 0 pA rms 500 MΩ Whole Cell 0 1 20 nA 1...

Страница 94: ... is generated at the input of the headstage and the cell sees this transient However after extensive tests on many types of cells in all recording configurations we have concluded that these switching transients are too small to cause any damage to the cell membrane I Clamp Mode In I Clamp mode Rf determines the maximum amount of current that can be injected into the cell without saturating the he...

Страница 95: ...eriments 3 dB Frequency The 3 dB or cutoff frequency fc of a filter is the frequency at which the output signal voltage or current is reduced to 1 2 i e 0 7071 of the input Equivalently fc is the frequency at which the output signal power is reduced to half of the input These terms arise from the definition of decibel dB Voltage dB 20 log Vout Vin Power dB 10 log Pout Pin For a low pass filter the...

Страница 96: ...ltiClamp 700A Bessel Filter This is the analog filter used for most signals for which minimum distortion in the time domain is required 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 Figure 4 10 Filter characteristics illustrated ...

Страница 97: ...a frequency greater than 1 kHz will appear in the digitized trace as extra noise in the range 0 to 1 kHz In other words higher frequency noise 1 kHz will appear under the alias of lower frequency noise 1 kHz This error is called aliasing A fundamental principle of signal analysis called the Nyquist Principle therefore states that in order to avoid aliasing the digitizing frequency fd should be at ...

Страница 98: ... minimally distorted by the filter According to the above equation then the appropriate filter setting would be 10 kHz In practice you may need to make other compromises For example if the signal is very noisy you may wish to filter more heavily and accept that the action potential risetime is artifactually slowed High pass Filter The Scaled Output and Scope signals can be high pass filtered by se...

Страница 99: ...he 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 Initially make only one connection to the MultiClamp 700A from the SCALED OUTPUT BNC to the oscilloscope After verifying that the hum levels are low start increasing ...

Страница 100: ...ect 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 ...

Страница 101: ...e interior of an electrode at a command potential while measuring the currents that flow down the electrode An I V converter achieves this by producing a voltage output that is proportional to the current input 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 ...

Страница 102: ...t A fundamental problem of this circuit when used for patch clamping is that the output bandwidth of the probe is inherently low To a first approximation the product of Rf and the stray capacitance sets the bandwidth across it For example if Rf is 500 MΩ and the stray capacitance is 0 5 pF the bandwidth is about 600 Hz To overcome this limitation the probe output is passed through a high frequency...

Страница 103: ...he resistance of the feedback resistor The rms current noise is given approximately by Irms 4kTfc Rf where fc is the filter cutoff frequency and k and T are constants Thus for low noise a high value of Rf is desirable This was pointed out in Chapter 5 FEEDBACK RESISTOR Current Clamp Circuit In I Clamp mode a separate headstage circuit is used called a voltage follower The essential features of a v...

Страница 104: ...ple 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 Commander Mounting the Headstage For maximum mechanical rigidity the CV 7A headstage should be mounted directly to the head of the micromanipulator using the dovetailed mounting plate Bath Connection The bath or ground electrode should be con...

Страница 105: ...wever if you are in a laboratory where static is high i e you hear and feel crackles when you touch things you should touch a grounded metal object immediately before touching the headstage Acoustic Pick up Rare cases have been reported in which the headstage was susceptible to low amplitude acoustic pick up For example the audible hum of a nearby isolation transformer can acoustically couple to t...

Страница 106: ...Help is accessed via the button at the top of the main MultiClamp Commander window In order for the On line Help to work properly the computer running the MultiClamp 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 ...

Страница 107: ...e needed clearance between the headstage case and other components in the experimental setup To further minimize the noise contributed by the holder in single channel recording the holder uses a small 1 mm pin for the electrical connection and a large amount of insulating Teflon Mechanical stability of the electrode is assured in several ways See Figure 4 13 As the pipette cap is closed the cone w...

Страница 108: ...se to pipettes with a large ID 1 1 mm Therefore the HL U is supplied with 0 25 mm silver wire which must be chlorided before use See below Spare components included with each holder are one 50 mm length of silver wire 40 cone washers 10 of each size and one 70 mm length of silicone tubing Cut into 2 mm lengths the silicone tubing will yield approximately 30 replacement silicone seals Additional co...

Страница 109: ... increase the noise Silver Chloriding It is up to you to chloride the end of this wire as required Chloriding procedures are contained in many electrophysiology texts1 Typically the chlorided wire will need to be replaced or rechlorided every few weeks A simple yet effective chloriding procedure is to clean the silver wire down to the bare metal using fine sand paper and immerse the cleaned wire i...

Страница 110: ...the silver wire insert the nonchlorided end through the hole of the silicone seal and bend the last 1 mm of wire over to an angle of 90 Press the wire into the back of the barrel making sure that the silicone seal is flush with the back of the barrel Slip the threaded collar over the back of the barrel With the large end of the pin directed toward the bent over wire screw the pin cap down firmly b...

Страница 111: ...gnal controlled by pCLAMP COMMAND Voltage or current commands to the MultiClamp 700A are accepted at this input The External Command Sensitivity is set in the 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 MultiClam...

Страница 112: ...urther amplified or filtered Headphone Jack This will drive headphones or a remote powered speaker if it is desired to monitor the audio output of the MultiClamp 700A The output is the same as that available at the rear panel AUDIO OUTPUT jack Rear Panel HEADSTAGE 1 2 The CV 7A headstages are plugged into the corresponding 25 pin DB connectors Note that Headstage 1 refers to Channel 1 inputs outpu...

Страница 113: ...V step aligned with the onset of the Seal Test Tuning or Pulse step 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 DEVICE ADDRESS This is used to specify the device number of the MultiClamp 700A The ...

Страница 114: ...is case a 100 pA step of current before the Output Gain is applied The Scaled Output signal will now be a flat line on which the single channel activity is superimposed This assumes that the capacitance transients at the start and end of the step have already been canceled using Capacitance Compensation Indeed Leak Subtraction can be thought of as a kind of capacitance compensation that applies to...

Страница 115: ...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 computer program like pCLAMP which allows off line leak correction ...

Страница 116: ... converter whereas current clamp is achieved with a voltage follower This contrasts with the design of other patch clamp amplifiers in which the same basic circuit is used for voltage clamp and current clamp producing a compromised performance The I 0 mode is a special case of I Clamp in which all external inputs are disconnected This is convenient if you wish to quickly return to the resting pote...

Страница 117: ...nstant is 16 5 ms and a 10 GΩ resistor models the patch The pipette capacitance is about 4 6 pF The charging time constant is approximately 330 µs 10 MΩ x 33 pF The PATCH 1U model cell has been made without a switch to change the model between the BATH PATCH and CELL positions This is because even the best switches have an enormous amount of leakage resistance and capacitance that increases the no...

Страница 118: ...106 Reference Section MultiClamp 700A Theory and Operation Copyright 2000 2001 Axon Instruments Inc Figure 4 15 PATCH 1U model cell ...

Страница 119: ...commonly used for quantifying white noise See Chapter 5 FILTERS Root mean square rms noise This is essentially the standard deviation of the noise and can be calculated using a computer or an electronic circuit designed for this purpose For white noise the rms noise is approximately one sixth the peak to peak noise The MultiClamp Commander displays the rms noise on the Membrane Current signal in V...

Страница 120: ...nce i e the electrode resistance plus input resistance of the cell Thus a low resistance electrode cell combination is preferred A large stray capacitance will reduce the noise by acting like an RC filter but this will also reduce the measurement bandwidth Increasing the Capacitance Neutralizaton setting will improve the bandwidth but increase the noise Electrode and Holder V Clamp Current noise i...

Страница 121: ...t noise because the compensation circuit employs positive feedback that injects noise back into the headstage Further the effect of Rs compensation is to reduce the electrode series resistance which reduces the effect of the RC filter mentioned above Electrode and Holder I Clamp Increasing Pipette Capacitance Neutralization increases the voltage noise for reasons similar to those just mentioned fo...

Страница 122: ...ovides 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 The Sync Output is a 0 to 3 4V step aligned with the onset of the Seal Test Tuning or Pulse step ...

Страница 123: ...ressing the button and then subtracting this value from all subsequent Scaled Output signals Unlike Leak Subtraction or Bridge Balance it does not account for currents or voltages that change as a result of time varying command pulses it simply provides a constant offset adjustment The Auto Output Zero only affects the signal on the Scaled Output In other words the cell is not affected by the Outp...

Страница 124: ...ting saturation in internal circuits The SCALED OUTPUT might not appear to be saturated because it may be heavily filtered reducing the size of any saturating transients at the output OVERLOAD reports any saturation 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 os...

Страница 125: ...potential outside the cell Vm Vin Vout Depolarization A positive shift in Vm e g from 60 mV to 80 mV caused by a flow of inward current Hyperpolarization A negative shift in Vm MultiClamp Polarity Conventions The conventions described here apply to all amplifiers manufactured by Axon Instruments To prevent confusion Axon always uses current and voltage conventions based on the instrument s perspec...

Страница 126: ...mple in the whole cell or outside out patch configuration the electrode tip is on the intracellular face of the cell Thus a negative potential Vp at the headstage input electrode interior is a negative potential inside the cell The cell s membrane potential under voltage clamp is therefore Vm Vin Vout Vp 0 Vcmd Positive current flowing out of the electrode must then flow from the inside to the out...

Страница 127: ...der counterclockwise 3 Replace the fuse with another fuse of the same rating 4 Reconnect the power cord Glitches The MultiClamp 700A 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 im...

Страница 128: ... Demo Mode This allows the MultiClamp Commander to be run without a MultiClamp 700A amplifier being connected or switched on Demo Mode is useful for exploring the features of the MultiClamp 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 700A is connected to a ...

Страница 129: ...h electrode usually minor Series resistance causes three major problems in voltage clamp recordings 1 Steady state voltage errors Suppose you are measuring a 1 nA membrane current under V Clamp If Rs 10 MΩ there will be a voltage drop of IRs 1 nA x 10 MΩ 10 mV across the series resistance Since Rs is interposed between the headstage and the cell membrane the actual cell membrane potential will be ...

Страница 130: ...Hz without Rs compensation Fortunately electronic techniques have been developed to partially correct for the errors caused by series resistance In V Clamp mode the techniques are generally referred to as Rs Compensation Series resistance errors can also occur in I Clamp mode These errors are generally corrected using the techniques of Bridge Balance and Capacitance Neutralization See these entrie...

Страница 131: ...nd 50 GΩ feedback resistors typically used for isolated patch recordings An exception is macropatches or nucleated outside out patches in which the currents can be quite large and for which Rs compensation may be necessary If Rs compensation is found not to be necessary it is best to turn it off This is because Rs compensation increases noise Adjusting Rs Compensation It is recommended that you pr...

Страница 132: ...ll controls See Chapter 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 4 16 Figures 4 15 4 19 Whole cell responses using PATCH 1U model cell with Rf 500 MΩ Scaled Output gain 10 and Seal Test 100 mV Figure 4 15 Uncompensated response with saturating ...

Страница 133: ...eference Section 121 Chapter 5 Figure 4 17 After setting Prediction 90 Correction 0 Figure 4 18 After setting Prediction 90 Correction 90 Figure 4 19 After optimizing Cf Rs and Cm to minimize transients ...

Страница 134: ...ent begin by reducing by a few percent the value of Rs MΩ displayed under Whole Cell As you reduce this setting the amplitude of the transient first decreases and then begins to increase A distinct minimum exists and the desired value of Rs is at this minimum Next slightly adjust the Cp Fast settings trying to further minimize any fast leading edge transients When this has been done small adjustme...

Страница 135: ...Whole Cell checkbox must be checked Whole cell compensation provides estimates of Rs and Cm which together determine the shape of the correction current that is injected through capacitor C2 Figure 4 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 bec...

Страница 136: ...ell 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 signal applied to the headstage capa...

Страница 137: ...ential 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 limitations imposed by the stability of the 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 ...

Страница 138: ...he complexity of this circuitry and a variety of non ideal characteristics cancellation of whole cell capacity transients does not remain perfect as Prediction is increased The small residual transient that emerges can however be completely removed by small readjustments of the setting of the Cp Fast and Whole Cell controls See Adjusting Rs Compensation above It should be noted that Prediction wou...

Страница 139: ...V At the same time the use of Correction will reduce the filtering effect of Rs and Cm on the measured current With 0 Correction the actual bandwidth of current measurement prior to any output filtering is limited to 1 2πRsCm which will be about 320 Hz in this example As Correction is increased this filter changes to 1 2πRsrcCm so that for 90 Correction the possible bandwidth for current measureme...

Страница 140: ...vertheless Prediction which is inherently stable up to 98 or more can be set to a value substantially higher than 70 about 95 thereby ensuring that the true transmembrane potential changes rapidly in response to the command potential even though a substantial series resistance remains uncompensated in terms of ionic currents Oscillations One of the practical problems when using the Correction func...

Страница 141: ...s normally the case for cells at rest containing small drug activated or synaptic currents However during voltage activation the cell membrane resistance could fall a hundredfold or more to values similar to or less than the series resistance In these cases it is probable that 1 There will be a significant error due to the voltage drop across the electrode This error is not obvious to the user bec...

Страница 142: ...amp 700A 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 Commander software Click on the Configure SoftPanel toolbar icon to access the menus for re configuring each knob or button After assigning the desired functions to each knob or button remove the pre defined ma...

Страница 143: ...being transmitted on the serial cable that connects the MultiClamp 700A to the host computer Under quiescent conditions the STATUS light flashes at about 2 Hz indicating that the MultiClamp Commander is interrogating the MultiClamp 700A in order to update its meter displays The STATUS light is useful for troubleshooting If it does not flash continuously a communication problem is indicated See TRO...

Страница 144: ... 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 use the minimum duration that is likely to achieve the desired result because too long a Zap could cause the seal resistance to deteriorate A duration of 0 5 or 1 ms is suggested for initial attempts Apply a repetit...

Страница 145: ...e MultiClamp 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 700A is not responding to commands The Status light is not flashing Possible causes The serial cable is not plugged in properly or is defective The PC s serial port is defective Select Device has been set...

Страница 146: ...ctrical continuity and DC stability of the electrode holder and bath electrode Check for bubbles in the microelectrode Check that the outside of the chamber is dry Set Pipette Capacitance Neutralization to zero Symptom Extraneous noise is present in the Scaled Output signal Pipette Offset is drifting rapidly Possible cause The Ag AgCl pellet or Ag wire in the electrode holder may be defective Dirt...

Страница 147: ...V 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 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 ...

Страница 148: ... ranges Slow capacitance compensation tau 10 µs to 10 ms in two ranges 10 200 µs and 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 5...

Страница 149: ... selectable from 0 to 10 V Rf amps at the electrode 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 ...

Страница 150: ...h frequencies Hz 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 One pole Bessel high pass filter with eight 3 dB cutoff frequencies Hz DC 0 1 1 3 10 30 100 300 External Inputs Command 20 mV V or 100 mV V sensitivity for V Clamp 400 pA ...

Страница 151: ... 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 The VCO ranges from 4000 Hz 100 mV to 300 Hz at 100 mV ...

Страница 152: ......

Страница 153: ...th 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 cell free membranes patches Pflügers Arch 391 85 100 1981 Joh...

Страница 154: ...le Channel Recording Second Edition Plenum Press New York 1995 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 ...

Страница 155: ...n shipping 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 ...

Страница 156: ...for old versions of software products will cease 12 months after they are upgraded or discontinued If you purchased your instrument from a Distributor or OEM Supplier contact them for repair service If you purchased your instrument from Axon Instruments contact our Technical Support Department If it is determined your instrument must return to the factory for repair the Technical Support Represent...

Страница 157: ...hich way is up We do NOT recommend using loose foam pellets to protect the MultiClamp 700A If the carton is dropped by the shipper there is a good chance that the instrument will shift within the loose pellet packing and be damaged 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 pr...

Страница 158: ...Clamp Capacitance Compensation 67 69 72 79 80 102 112 117 118 119 Model Cell 14 105 119 Whole cell 14 39 70 Electrochemistry 77 External Command 67 72 79 80 81 82 83 86 99 External Command Inputs 67 72 79 81 83 86 99 Filter 79 80 84 85 86 119 122 136 138 Functional Checkout 6 Gain 20 100 102 112 135 138 139 Glass Dimensions 135 Types 84 Grounding 87 107 109 115 Headstage Adapters 98 Cleaning iii 9...

Страница 159: ...e Selection iii 115 Power Supply Glitches 115 Practical Guide Forming a Gigaseal 36 Interfacing a Computer 34 Low Noise Techniques 36 43 Perforated patch Recording 42 Sharp Microelectrode Recording 35 47 73 75 108 Whole cell Voltage Clamp Recording 39 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 Scaled Output ...

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