Oocyte Clamp Amplifier User`s Manual
Contents
1. command voltage Acquisition and display of data is also usually handled by computer The OC 725C is fully compatible with all commercially available software packages designed for electrophysiological research Finally a microelectrode puller is necessary for making appropriately sized voltage and current electrodes Usually the microinjection pipette puller can also be used to make microelectrodes You will need use of a micro scope to break off the pipette tips Publication 5720 001 REV B J Appendix Specifications Voltage recording channel V Oocyte Clamp Amplifier Model OC 725C Warner Instruments Input Impedance Output Resistance V n OFFSET Noise Electrode Test Negative Capacity V Meter Range full scale Bath electrode channel I V OFFSET Noise Clamp clamp on Open loop clamp off MONITOR MONITOR FILTERED 4 pole Bessel Gain Telegraph Meter Ranges full scale V clamp off x0 1 range x1 0 range x10 range Current electrode channel V Compliance Voltage Alarm 160 V Gain Variable DC Electrode Test Commands Hold internal External input attenuated by 10 Maximum external input Power requirements Dimensions Enclosure Voltage Headstage Mounting Handle Bath Headstage Publication 5720 001 REV B 5 x10 Q shunted by 3 pF 100 Q 200 mV at V probe input 50 mV RMS at 1 kHz 10 mV MQ 0 45 pF 199 9 mV 200 mV 5 5 nA R2. membrane potential should be mounted on a micro manipulator and con nected to the VOLTAGE PROBE socket VOLTAGE ELECTRODE section High voltage current electrode The holder should be mounted on a micro manipulator and the cable connected to the I ELECTRODE socket CURRENT ELECTRODE section Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup Cont d Command potential If a computer or external generator is used for con trolling the clamp command potential its signal should be connected to the front panel COMMAND IN 10 input COMMANDS section External monitoring To monitor the microelectrode s potentials on an oscilloscope computer or a chart recorder the following connections should be made V Membrane potential may be recorded from the V x10 connector VOLTAGE ELECTRODE section V e The voltage of the current electrode can be monitored from the V con nector on the REAR PANEL The output will be the same as that reported on the CURRENT ELECTRODE METER showing the voltage across the current electrode when the CLAMP SELECTOR Switch is in the off position Recall that the meter indicates the current d D when the CLAMP SELECTOR switch is in either the slow or fast position I The current signal is available from the 1 MONITOR and I MONITOR FILTERED outputs BATH ELECTRODES section Gain telegraph Automatic monitoring of the I gain can be achieved by con
3. output from your computer DAC to the COMMAND IN 10 BNC connector See Cable Connections page 20 17 Depending on the amplitude of the response you wish to record you may adjust the instrument GAIN to a higher or lower position The CURRENT ELECTRODE METER should now be displaying the current in uA that is delivered to hold the cell at the designated command potential Clamping high conductance cells 18 Dc GAIN mode CLAMP section may be required to clamp high con ductance low resistance cells This condition will be evidenced by the inability of the instrument to maintain a DC holding potential to within 196 or better of the set value and the maximum instrument gain is not sufficient to provide a hard clamp DC GAIN mode pro vides an additional DC gain greater than 105 while the AC gain remains at 2000 maximum for stability Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments A Procedure for Recording from Oocytes Unclamping the cell 19 To unclamp the cell turn the GAIN control CLAMP section fully count er clockwise to the detent off position This will also disengage the pc GAIN NOTE If the control is not fully off and the Dc GAIN is left on the reparation will not be unclamped Removing the electrodes 20 It is very important that the CLAMP GAIN be returned to the off posi tion fully counter clockwise to click off as described above and the CLAMP MODE selector
4. slow The slow clamp mode is useful for screening of oocytes or where high clamp speeds are not required The slow clamp speed is approximately 0 5 ms when measured with the model membrane 1 MO shunted with 0 47 uF In this mode measured currents are displayed on the BATH ELECTRODES METER in units of uA fast Most oocyte clamping is performed in the fast mode The clamp speed is limited by the resistance of the current electrode and the oocyte membrane capacitance Therefore the current electrode resistance must be kept as low as possible to obtain the fastest clamp speeds Currents are read on the BATH ELECTRODES METER in units of uA Fast clamp speeds are 350 us when measured with the model cell as described above The GAIN control is a single turn potentiometer which varies the full bandwidth open loop gain from 0 to 2000 A high pc GAIN 105 can be switched in with the pc GAIN toggle switch to provide a hard clamp when passing large currents from high expression oocytes Publication 5720 001 REV B S Oocyte Clamp Amplifier Model OC 725C Warner Instruments Control Description Conto Commands The COMMANDS control block contains the HOLD COMMANDS controls and COMMAND IN 10 input BNC HOLD controls HOLD potential is set with the DIGITAL POTENTIOMETER thumbwheel and RANGE toggle switch Ranges are 99 mV and 198 mV depending on the scale multiplier selected x1 0 or x2 0 Signal polarity or off is selected wi
5. switch be placed in the off position before removing the current electrode from the cell Failure to perform the above steps will overload the feedback ampli fier due to the large current generated when the membrane resist ance between the current electrode and the bath virtual ground goes to zero This will damage the oocyte For this reason we recommend that the user enable the audible overload alarm to provide a warning when the potential for such damage exists Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Special Circumstances High side current measuring In studies of single oocytes current is monitored by the bath clamp head stage Experiments involving two oocytes in a common bath such as gap junction studies requires monitoring currents from each oocyte This is done in the current output leg Gin series with and ahead of the current elec trode Two disadvantages of monitoring the current in this manner exist a The noise level of this signal is higher However this is usually not a serious problem since currents are typically in the uA range b The voltage drop across the solution resistance from oocyte to bath ground becomes an error voltage since it is not subtracted out as when the bath clamp headstage is used This problem is overcome by using the optional DIFFERENTIAL VOLTAGE HEADSTAGE Configuration The OC 725C current measuring circuit can be changed t
6. GAIN SELECT BATH ELECTRODES x40 CLAMP MODE switch CLAMP off DC GAIN toggle CLAMP out GAIN CLAMP CCW to detent off HOLD POTENTIAL COMMANDS 00 mV HOLD POTENTIAL MULTIPLIER COMMANDS x1 0 HOLD POLARITY COMMANDS off V OFFSET CURRENT Center of rotation ELECTRODE approximately 5 turns Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Using the Model Membrane cont d Test procedures In the following testing procedures allow a tolerance of 1 on the read ings taken For example if the test response is indicated as 100 mV a read ing from 99 0 to 101 0 mV would be within tolerance Offset controls V OFFSET VOLTAGE ELECTRODE section The full range of this control is 200 mV This can be verified by rotating the control first fully clock wise and then fully counterclockwise while observing the VOLTAGE ELECTRODE METER The displayed readings will indicate off scale at the extremes of the control s manipulation since the meter is only capa ble of displaying 199 9 mV Vi X10 output BNC VOLTAGE ELECTRODE section This output can be monitored using an oscilloscope The reported voltage will swing between 2 V as the V OFFSET control is manipulated throughout its full range Set the V OFFSET to 0 0 reading on the meter and verify that the V x10 reading on the scope also reads 0 V V OFFSET Control CURRENT ELECTRODE section The V OFFSET
7. MS at 1 kHz x1 range 28 pA RMS at 1 kHz x1 range 0 01 100 V mA in 3 ranges 7 steps per range Same as above filtered at 1 kHz 0 2 2 6 VDC in 0 2 V steps 199 9 mV 199 9 uA 19 99 uA 1 999 pA 180V 0 2000 AC DC 1 x10 DC switch selected 10 mV MQ 198 mV in 2 ranges 1 V in 0 1 V command 10V 100 130 or 220 240 VAC 50 60 Hz 9 x 42 x 25 cm H x W x D 1 25 x 5 cm dia x length with 1 8 m cable 4 8 mm x 6 3 cm dia x length 2 8 x 3 5 x 4 2 cm H x W x DJ with 1 8 m cable All noise measurements made with an 8 pole Bessel filter Oocyte Clamp Amplifier Model OC 725C Warner Instruments Appendix Conto Gain telegraph outputs I Output V pA Gain select settings Gain select Headstage resistor I output range V A Gain Telegraph I max output m A Maximum meter reading x0 1 10 kQ 0 01 1 0 10 1000 199 9 pA x1 0 100 kQ 0 1 10 1 100 19 99 pA x10 1 MQ 1 0 100 0 1 10 Noise from bath feedback resistor Noise 1 999 uA Feedback Resistor Shorted Input Standard Model Cell 0 5 pF Modified Model Cell 0 22 pF 10 KQ 75 pA 6 0 nA 4 4nA 100 KQ 28 pA 5 5 nA 4 4 nA 1 MQ 22 pA Publication 5720 001 REV B 5 0 nA 4 0 nA E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Appendix Conto References Co
8. Oocyte Clamp Amplifier Model OC 725C WARNER INSTRUMENTS A Harvard Apparatus Company Publication 5720 001 REV B WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of our products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive We will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as t
9. al voltage headstage is available which subtracts the voltage drop across the series resistance in the bath Voltage Headstage Probe The voltage measuring headstage is a single ended high impedance probe Its small size convenient mounting rod and two meter cable make for easy attachment to a micropositioner A supplied electrode holder with a 2 mm jack mounts directly on the headstage Voltage and Current Meters Independent meters provide simultaneous displays of membrane voltage V and membrane current I To assure prop er impalement of the current electrode the current meter displays mem brane potential V from the current electrode before the clamp circuit is activated Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Introduction conta Additional Features Buzz controls for each electrode aid in penetration of cell membranes with a minimum of leakage Electrode Test for voltage and current electrodes Capacity Compensation for the V voltage input Overload Alarm serves as a reminder when the feedback amplifier reach es its maximum output voltage a condition which could result in damage to the oocyte DC Offsets for voltage and current electrodes Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Nomenclature Text conventions This manual refers to amplifier controls at four functional levels opera tional sectio
10. cation of Council Directive 73 23 EEC Standards To Which Conformity Is Declared Manufacturer s Name Manufacturer s Address Equipment Description Equipment Class Model Numbers EN 61010 1 1993 Warner Instrument Corp 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Heater Controller Safety requirements for electrical equipment for measurement and laboratory use Class I TC 324B and TC 344B I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standards Publication 5720 001 REV B Place Hamden Connecticut USA Signature Ma A tat Full Name Ralph Abate Position Business Manager
11. control is tested in the same manner as the V OFFSET control This control is adjustable when the CLAMP MODE switch is set to off The voltage of the V OFFSET is read from the BATH ELECTRODE METER when the CLAMP MODE Switch is set to off V x10 output BNC This BNC is located on the rear panel of the instrument and reports the setting of the V orrser control when the CLAMP MODE switch is set to off Return all controls to their initial settings when done with this test Voltage electrode test In actual practice the voltage electrode test is used prior to entering the cell and indicates the resistance of the electrode When used in conjunction with the model cell it measures both the electrode and membrane resistance 2 MQ This test is performed using controls in the VOLTAGE ELECTRODE section With the model cell in place depress the V ELECTRODE TEST pushbut ton and observe a reading of 20 mV on the VOLTAGE ELECTRODE METER This corresponds to a 2 MQ reading at a calibrated response of 10 mV MQ On the oscilloscope the V x10 output will read 200 mV which is X10 the applied test voltage Since the test current is being passed through 1 MO R the I MONITOR output will indicate 1 mV which corresponds to 10 nA of current Publication 5720 001 REV B i Oocyte Clamp Amplifier Model OC 725C Warner Instruments Using the Model Membrane cont d With the CLAMP MODE switch set to off the BATH ELECTRODE METER mo
12. crew mounting slots is attached to the probe base The BATH PROBE connects to the control unit with a 6 pin connector Current electrode cable A two meter shielded cable is sup plied with a 2 mm pin jack on one end to mate with the supplied P electrode holder and a 3 pin con nector on the other end to mate with the instrument The electrode holder has a handle for mounting in a micromanipulator Model cell The model cell supplied with the OC 725C is useful as a training aid and as a calibration and test device It has connections for the voltage and current probes and to the bath clamp allowing all aspects of the amplifier s function to be tested Comments Connecting to line power The model OC 725C is supplied with a 3 conductor power cord One conductor provides a connection between the instrument housing and the earth ground Safe operation of the instrument will be assured provided that the ground circuit in the power outlet is wired correctly and is connected to earth Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Control Description Conto NOTE If the ground pin of the power cord is removed for any rea son the instrument chassis must be directly connected to earth ground using a separate heavy 14 gauge or larger ground wire High voltage outputs When handling the current electrode cable be sure to set the GAIN CONTROL fully clockwise and the CLAMP MODE s
13. cubation The less damage done to the mem brane during handling and impaling the cell the happier it will be Use of the BUZZ function should help minimize the trauma from electrode pene tration Membrane damage can be further reduced by properly isolating the experimental platform from vibration Finally hydraulically driven micro manipulators will also reduce membrane damage while the electrodes are in the cell Repeated recordings Most recording sessions will involve repeating the above steps several times with many different cells Unless there is a concern about contami nation of the bath solution by something carried over from previous exper iments the pipettes can also be used repeatedly They should be free of debris and should have approximately the same resistance as they had in the previous recording A significantly higher resistance could indicate that the pipette is partially plugged with cellular debris Make the following control settings before the next recording is carried Control Section POLARITY COMMAND off MODE SELECT CLAMP off GAIN CLAMP 0 fully CCW Electrophysiology If you are well versed in setting up electrophysiological equipment you can safely skip over the rest of this section If however this is your initiation into electrophysiology as it may well be for some of you molecular biolo gists then you may find the following recommendations helpful While the whole cell elec
14. der assembly is mounted in a micropositioner with the mounting rod supplied Bath probe The bath clamp is designed to maintain a virtual ground in the oocyte per fusate The bath probe should be positioned so that the silver electrode wires can be inserted into the recording chamber or into the agar bridge wells Sticky wax or tape is usually sufficient to secure the unit when posi tioned on a flat surface or alternatively the unit can be held in place on a separate stand The bath probe electrodes should also be chlorided before use as described above Electrode placement and grounding Three drawings shown on pages 19 and 20 have been included to illus trate the various ways a bath circuit can be configured Most applications involve only a single oocyte and Figures A and B illustrate these setups Figure C shows a setup for recording from 2 oocytes in a common bath with the use of dual clamps NOTE Cable routing must be performed with care Bundle cables together rather than routing them individually and keep them as far as possible from sources of 50 60 Hz interference e g line cords transformers etc Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup Cont d Bath clamp electrode placement Proper placement of the bath electrodes Qo and Lense is important for obtaining optimum performance The I electrode or the agar bridge associated with it sh
15. dstages 7250V PROBE Standard Version The voltage probe is an active headstage housed in a 1 25 x 5 cm cylinder dia x length The probe body is nickel plated and epoxy sealed for corrosion resistance NOTE The outer shell is electrically driven at the input potential The supplied microelectrode holder mates directly to the 2 mm input pin on the probe body A mounting block and handle are also supplied and facilitate attachment of the probe to a micromanipula tor The handle can be mounted either axially or perpendicular to the probe body Publication 5720 001 REV B 5 Oocyte Clamp Amplifier Model OC 725C Warner Instruments Control Description Conto 7255DI DIFFERENTIAL PROBE Optional This voltage probe is designed for applications where two oocytes share a common bath or where the voltage drop across the solution resistance is to be measured and subtracted from V The headstage housing is approximately 2 cm longer than that of the 7250V PROBE and has two additional inputs CIRCUIT GROUGN and V DIFFERENTIAL When the two inputs are shorted the probe functions exactly the same as the standard single ended 7250 PROBE Bath headstage The BATH PROBE is housed in a 2 8 x 3 5 x 4 2 cm aluminum enclosure Inputs are two 1 mm pin jacks labeled I sENsE and I ovr The case is electrically grounded and a pin jack is located on the side for con necting to shields A plastic plate with two s
16. e voltage electrode The VOLTAGE ELECTRODE METER Will display the resulting potential in mV The resistance of the electrode can be easily calculat ed by dividing the current into the potential The resulting answer will be expressed in Q For example if the electrode test indicates that a potential of 25 0 mV is produced by the 10 nA test current _ X 25mV I 10nA 2 5x10 Q 2 5MQ NOTES a A simpler calculation is to divide the voltage readout by 10 and append the units of MQ e g 25 mV 2 5 MO b The calculated resistance value may vary widely from pipette to pipette but should be less than 4 MO for the voltage electrode Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments A Procedure for Recording from Oocytes Current electrode placement 7 8 9 Advance the current electrode until the tip is in the chamber bath solution Adjust V OFFSET for a zero reading on the CURRENT ELECTRODE METER This will establish a null reference allowing the resting poten tial to be directly read With the CLAMP SELECTOR switch in the off position the resistance of the CURRENT ELECTRODE pipette is tested in the same manner as the VOLTAGE ELECTRODE Pressing the V ELECTRODE TEST pushbutton will cause a 10 nA current to be passed across the CURRENT ELECTRODE The resulting voltage in mV will be displayed on the METER in the BATH ELECTRODE section From that value the resistance of t
17. ed from the voltage electrode and that shield should be grounded to the circuit ground This can be accomplished by wrapping the current pipette with aluminum foil or by mounting a metal screen or plate between the two pipettes In either configuration the shield can be grounded by connecting it to the ground mini jack on the side of the bath probe When using the aluminum foil method care must be taken to prevent the foil from touching the sur face of the bath solution at the bottom end of the pipette or the silver elec trode wire at the top end Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup Cont d Electrode holders Voltage Electrode The voltage recording electrode holder uses a silver wire for the electrical coupling between the pipette and holder connector Any silver wire contacting the KCl solution in the pipette must be chlorided to reduce junction potentials see Chloriding Procedure in Appendix The pipette should contain just enough KCI so that approximately 1 2 inch of the chlorided wire is submerged The pipette holder assembly is attached directly to the voltage headstage prior to mounting in a micropositioner Current Electrode The electrode holder supplied for the current electrode also uses a silver wire for coupling In an manner analogous to the voltage electrode the current electrode wire must also be chlorided prior to assem bly and use The pipette hol
18. en 27 REMOVING Ihe electrodes ona aseo nente oie 27 Special Circumstances ssseseeeeenennententnta nnns 28 High side current measuring 28 refe rfe reco o M 28 Optional voltage headstage se 28 Comments and Recommendations sss 29 30 Membrane damage senes 29 Repeated recordings 1 neret 29 Electrophysiology reete 29 Dpc 31 35 Specifications Equipment is intended to be operated in a controlled laboratory environment essere 31 Gain telegraph outputs seen 32 Gain select settings sse 32 Noise from bath clamp feedback resistor 32 References tette tette nettes 33 Certifications iuiar oaii 34 Certifications tenentes 35 Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Introduction The model OC 725C Oocyte clamp is designed for two electrode whole cell voltage clamping of Xenopus oocytes as well as for other large cells and cell structures such as squid axons The instrument has several features mak ing it ideal for these purposes Unique Features High Voltage Compliance The OC 725C combines high AC and DC gains and a voltage compliance of 180 V to insure fast nonsaturating clamp petr formance
19. ent settings 13 Test procedures dando antennal 14 Offset CONT OMS occid n e etel det 14 Voltage electrode test ntt 14 r M 15 Current electrode fest iiec 15 DC clamp tOSt einsi 15 AG Clamp IESi eim o EI tee RE 16 Oocyte Clamp Amplifier Model OC 725C Warner Instruments Table of Contents conto oE Pipettes eiie m rer eter anit tint haere us Electrode holders sss ZEE EE Electrode placement and grounding sses Bath clamp electrode placement seen Single oocyte setup with indirect ground Single oocyte setup with direct ground Dual ooctye Set up siirinsesi Cable connections sse Resting position of the controls Using the gain select seeenn Other gain range selection considerations 23 A Procedure for Recording from Oocytes 24 27 Initial electrode placement e 24 Voltage electrode placement sss 24 Current electrode placement sse 25 Imipaling he Cell ioaaonasonn taimiuninatatertatiaataines aie 25 Clamping the cell eene 26 Clamping high conductance cells es 26 Unclamping the cell e
20. et the HOLD POTENTIAL COMMANDS section to 100 mV 50 mV on thumbwheel and MULTIPLIER toggle at x2 Select positive pos polari ty The VOLTAGE ELECTRODE METER should read 100 mV and the CURRENT ELECTRODE METER should read 0 00 uA Switch to negative neg polarity The VOLTAGE ELECTRODE METER should read 100 mV and the CURRENT ELECTRODE METER should read 0 2 uA Return the GAINE control fully CCW and turn the CLAMP MODE switch to off Publication 5720 001 REV B 7 Oocyte Clamp Amplifier Model OC 725C Warner Instruments Using the Model Membrane conto AC clamp test Adjust the Vm orrsET control to 0 0 V Apply a 0 8 V 100 Hz square wave to the COMMAND IN 10 BNC Monitor the Vm x10 and I MONITOR outputs on the oscilloscope Switch the CLAMP MODE switch to fast and increase the GaIN until V reads 80 mV Verify that the V x10 BNC reports 0 8 V As you further increase the GAIN control you will see the rise time of the oscillo scope trace become faster since the speed of the clamp is limited by the resistance of the current electrode and the capacitance of the oocyte If ringing oscillation is observed decrease the GAIN setting to obtain the fastest clean waveform as shown below The current signal 1 MONITOR shown in the figure below displays the high cur rent spikes required to charge the oocyte capacitance Set the HOLD control to a reading of 50 mV and switch the POLARITY toggle to pos The square wave will be d
21. g membrane damage by elec trodes is to enhance the possibility of making subsequent recordings from the same cell These suggestions are also important for minimizing mechan ical noise in the recorded data You will need a dissecting scope for viewing the placement of the elec trodes Anything more powerful than 40x will just get in the way The light source for your scope should be DC and may need to be IR and UV filtered if you plan to use it during recording Minimally the recording chamber can be a stable surface on which the oocyte will not roll around A disposable petri dish with a piece of nylon mesh on the bottom has been successfully used for this purpose The dish can be stabilized by a holder or by some wax placed around its perimeter The diameter of the dish needs to be large enough to accommodate the oocyte and the two bath electrodes The walls of the recording chamber should be low enough to not interfere with electrode placement Perfusion of the chamber can be accomplished using a gravity fed system Perfusate from the dish can be evacuated using gentle vacuum Use as small an aper ture as possible to avoid disturbing the surface of the perfusate in the dish NOTE Specialized equipment suitable for oocyte studies oocyte chambers perfusion systems a Faraday cage a noise free DC lamp and micro manipulators are available from Warner Instruments Although it is not required a computer can be employed to control the
22. he pipette can be calculated exactly as described above i e divide the readout by 10 to get the resistance in MO Since the current electrode has a larger diameter its resistance should be less than that of the voltage electrode about 1 0 MQ or less If no voltage display is present during the electrode test procedure and you re sure that the electrode is contacting the bath perform the following checks a Make sure that all cables are connected properly b Check to see that the aluminum shield around the current electrode pipette if used is not touching the bath solution or the electrode wire c Check the current electrode to see if there is a bubble in the pipette causing an open circuit Impaling the cell 10 Recheck the VOLTAGE ELECTRODE METER to verify that the potential is correctly offset to read 0 0 mV and readjust the V OFFSET control if needed 11 Advance the VOLTAGE ELECTRODE until its tip is slightly depressing the plasma membrane of the cell and depress the V Buzz pushbutton This will produce a 1 V 1 kHz oscillation at the voltage electrode dis rupting the membrane and causing the tip of the electrode to impale the cell with no further movement of the micro manipulator this technique will work best with fresh oocytes i e 1 or 2 days post excision If the buzz technique fails to cause electrode penetration further advance the voltage electrode until it pops through the membrane P
23. hey apply Do Not Dispose Product with Municipal Waste Special Collection Disposal Required E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Table of Contents IntrOQU COR cierres tristia e anra ates tin ici peas Fear or C Race RC Unique Features High Voltage Compliance Bath Clamp Headslage n tte een Dual Oocyle Studles or ertt etd cR Voltage Headstage Probe Voltage and Current Meters Additional Features essent Buzz controls Electrode Test ai Capacity COMPCNSAION 2 itti n t tier ead Overload Alarm ni ti rep UBER UR ER REEL ARa pejor ET Ubrum kinane Text conventions Control Description ETOFIE DANG recenset etre tertiae nie teorie Voltage electrode ttti 6 Batlielectr d6s itte E Reis 7 Clamp section a COMMAS teste cas znai aa Aa 9 C rrent el ctiod6 i trt rp it aad 9 near DAMON ss ox soos pcc c t i 10 Additional components sene 10 Voltage recording headstages seen 10 Bath headslage b rm HR 11 Current electrode cable sse 11 Model Cell ortam Dudum qutd uu Ecce 11 Comments mall Connecting 10 line DOWOE 5 catre ertet tes 11 High voltage outpuls neant Lr e eise peo 12 Using the Model Membrane enne 13 16 Initial instrum
24. isplaced 50 mV in the posi tive direction Switching the polarity to neg will produce a 50 mV offset mV Beesseoxneees 10 8 6 4 2 0 2 4 6 8 10 Publication 5720 001 REV B 4 Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup The following instructions are designed to guide the user step by step through a typical recording session involving a Xenopus oocyte It is assumed that the user is already familiar with the techniques of Xenopus oocyte excision and microinjection for a review of those techniques see Colman 1984 It is also assumed that the user has some familiarity with the basic circuitry of a two electrode voltage clamp for review see Hille 1984 Pipettes Microelectrodes can be made using the same glass tubing and dimensions as those used for a typical patch pipette and are usually filled with 3 M ster ile filtered KCl Unlike the pipettes used as patching electrodes microelec trode pipettes do not require fire polishing nor coating with Sylgard They will need to be broken off however to a relatively large diameter to insure a fast response time by the clamp For the voltage electrode the pipette tip should be broken back to an O D of 3 5 um The current electrode pipette should be broken back to an O D of 7 9 um The resistances of these pipettes should be about 2 MQ and 1 MQ or less respectively When installed the current electrode pipette should be shield
25. l OC 725C Warner Instruments Control Description Conto Rear panel The line power connector and fuse are located on the rear panel Operating voltage is specified on the MODEL SERIAL NUMBER sticker applied to the rear of the instrument The rear panel also contains V x10 and GAIN TELEGRAPH Out put BNCs the ALARM switch and instrument GROUNDS NOTE Verify that the instrument is wired for the proper voltage before connecting the line cord The V x10 output BNC monitors the voltage of the current electrode x10 when the CLAMP MODE SELECTOR switch is off The GAIN TELEGRAPH Output BNC provides a DC voltage indicating the gain setting of the instrument The output varies from 0 2 to 2 6 volts in 200 mV steps as shown in the appendix ALARM switch Activates or deactivates the OVER VOLTAGE current elec trode audible alarm GROUNDS Both circuit and chassis grounding posts are located on the rear panel CHassis is common with the instrument enclosure and con nected to earth through the power line cord A shorting link allows for interconnection of the two grounds In most experimental setups separating the grounds will result in minimizing 50 60 Hz signal interference from ground loops However trial and error will deter mine the best results NOTE For safe operation the ground pin on the power plug must not be removed and the use of cheater plugs must be avoided Additional components Voltage recording hea
26. le larger currents without saturating important when recording from high expression oocytes Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments A Procedure for Recording from Oocytes Initial electrode placement 1 Make sure that the bath electrodes are submerged in the chamber or 2 in the agar bridge wells with the agar bridges completing the circuit to the bath and the oocyte is stable on the chamber floor Install the voltage and current pipettes onto their respective holders but do not yet place them in the chamber bath solution Voltage electrode placement 3 4 5 6 Advance the voltage recording electrode into the bath The VOLTAGE ELECTRODE METER will indicate in mV the potential between the elec trode and the bath If there is no voltage reading and you are sure that the pipette tip is in the bath solution perform the following checks a make sure that all cables are connected properly b inspect the voltage electrode to see if there is a bubble in the pipette which will cause an open circuit Using the V4 OFFSET control adjust the VOLTAGE ELECTRODE potential to read 0 mV If the junction potential of the voltage electrode can not be adjusted to 0 mV the electrode holder may be at fault See Electrode Holders page 17 To test the resistance of the voltage electrode pipette depress the ELECTRODE TEST button This passes a 10 nA current across th
27. lectrode holder the current sensing bath probe with silver wire electrodes and the current cable with electrode holder Front panel The instrument front panel is divided into six control blocks titled VOLTAGE ELECTRODE V BATH ELECTRODES L CLAMP COMMANDS and CURRENT ELEC TRODE The instrument rear panel has BNC connectors for GAIN TELEGRAPH OUTPUT and V x10 output an ALARM 077 0ff switch and binding posts for CIR CUIT and CHASSIS GROUND Voltage electrode The VOLTAGE ELECTRODE control block contains the VOLTAGE PROBE CONNECTOR the VOLTAGE METER the V OFFSET control the ELEC TRODE TEST and BUZZ push buttons and the V OUT PUT BNC Controls for NEGA TIVE CAPACITY COMPENSATION are also located in this block The VOLTAGE METER reports the membrane voltage V with a full scale range of 199 9 mV The VOLTAGE PROBE CONNECTOR is a 7 pin DIN connector for attachment of the voltage probe to the instrument The V OFFSET control is a 10 turn potentiometer providing up to 200 mV at the VOLTAGE PROBE input for offset of membrane junction potentials The V ELECTRODE TEST control is used to determine the internal resist ance of the voltage probe When the pushbutton is depressed a con stant 10 nA current is passed through the voltage electrode produc ing a voltage drop of 10 mV MQ of probe resistance The measured Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Wa
28. lman A 1984 Translation of eukaryotic messenger RNA in Xenopus oocytes Transcription and Translation eds B D Hames and S J Higgins IRL Press Oxford Ch 10 Hille B 1984 Ionic Channels of Excitable Membranes Sinauer Sunderland MA Ch 2 Zhou J Potts J E Trimmer J S Agnew W S and Sigworth EJ 1991 Multiple gating modes of the uI sodium channel Neuron 7 775 785 Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Appendix CE Certifications EMC Declaration of Conformity CE MARKING EMC Application of Council Directive 89 336 EEC Standards To Which Conformity Is Declared Manufacturer s Name Manufacturer s Address Equipment Description Equipment Class Model Numbers EN55022 Class A EN61000 3 2 EN61000 3 3 EN50082 1 1992 EN61000 4 2 EN61000 4 3 ENV50204 EN610000 4 4 EN610000 4 8 EN610000 4 11 Warner Instrument Corp 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Heater Controller ITE Class A TC 324B and TC 344B I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Publication 5720 001 REV B Place Hamden Connecticut USA Signature Full Name Ralph Abate Position Business Manager Oocyte Clamp Amplifier Model OC 725C Warner Instruments Appendix Conto LVD Declaration of Conformity CE MARKING LVD Appli
29. n current is read from the high side of the current output leg This method also requires the use of the optional DIFFERENTIAL VOLTAGE HEADSTAGE Two disadvantages are 1 The noise levels of the current Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup Cont d signal measured in the high side is approximately double that obtained with a bath clamp and 2 high levels of clamp current could produce a substantial voltage drop across the solution series resistance NOTES The oocyte clamp has no provisions for compensating the voltage drop described above The differential voltage electrode should be placed close to the oocyte and in the current path between the Vm electrode and ground Dual ooctye set up Dual oocyte setup is accomplished using two clamps as illustrated in Figure C Both clamps must be configured to read current from the high side as described in the section titled HIGH SIDE CURRENT MEASURING see page 26 and each clamp must be equipped with the optional DIFFERENTIAL VOLTAGE HEADSTAGE CLAMP1 A 477 BATH RESSTANCE NA x m NA V DIFF ELECTRODE 1 V N AN VOFF ELECTRODE 2 V JN BATH REFERENCE ELECTRODE Figure C Cable connections Bath clamp headstage After positioning the probe as described above con nect it to the BATH PROBE socket BATH ELECTRODES section Voltage electrode headstage The high impedance probe for recording
30. necting the rear panel GAINE TELE BNC to the appropriate input on your analog to digital converter Power The power cord should be connected to a properly grounded AC receptacle with the line voltage specified on the instrument nameplate REAR PANEL Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup Cont d Resting position of the controls To begin set the instrument controls to the following positions Control Section POWER I MONITOR Output BATH ELECTRODE GAIN SELECT BATH ELECTRODE HOLD COMMANDS POLARITY toggle COMMANDS MODE SELECT CLAMP CLAMP DC GAIN toggle CLAMP out CLAMP GAIN CLAMP off fully CCW ALARM REAR PANEL off ot on as desired Turn POWER On Using the gain select A wider range of bath current Q measurements is now possible with the addition of the GAIN SELECT toggle switch located above the GAIN control The switch has 3 positions x0 1 x1 and x10 Resistance values shown below each LED indicate the bath clamp feedback resistor used for the current measurement The chart on the next page shows the effect of the range selection on the I output Note also that range selection changes the sensitivity of the cur rent meter Typically measured currents will fall in the x7 range The lower and higher ranges are intended to cover those applications where currents are beyond the x range Currents below 1 uA should be m
31. ni tors V voltage at the current electrode In this case V will be a measure of the voltage across R and the meter will indicate 10 mV 1 MO The V x10 output BNC on the rear panel can also be checked to see that it reads 100 mV meter reading x10 Buzz This test is performed using controls in the VOLTAGE ELECTRODE section Set the oscilloscope sensitivity to 5 V div and depress the Buzz push button while monitoring the V x10 output AlkHz square wave of approximately 24 V p p will be generated as long as the button is depressed Current electrode test This test is performed using controls in the CURRENT ELECTRODE section With the model cell in place depress the V ELECTRODE TEST pushbut ton and observe a reading of 20 mV on the CURRENT ELECTRODE METER This corresponds to a 2 MQ reading at a calibrated response of 10 mV MQ With the CLAMP MODE switch set to off the BATH ELECTRODE METER moni tors V voltage at the current electrode In this case V will be a measure of the voltage across R and the meter will indicate 10 mV 1 MO DC clamp test This test is performed using controls in the CLAMP ELECTRODE Section Place the CLAMP MODE switch in the fast position and adjust the V OFFSET control for a reading of 100 mV on the VOLTAGE ELECTRODE METER Turn the GAIN control on and slowly turn the control clockwise until the meter reading V decreases to zero The CURRENT ELECTRODE METER should read 0 10 uA S
32. ns control blocks specific controls within a block and settings of specific controls To minimize the potential for confusion we have employed several text conventions which are specified below Since our goal is to provide clarity rather than complexity we welcome any feedback you may wish to provide e Warner Instrument product numbers are presented using a bold type References to instrument panel control blocks are specified using UNDERLINED SMALL CAPS g COMMANDS CLAMP References to specific controls within a block are specified using NON UNDERLINED SMALL CAPS g MODE SWITCH DC GAIN Finally references to individual control settings are specified in italic type e g slow fast 100 mV Special comments and warnings are presented iniiis Any other formatting should be apparent from context THIS EQUIPMENT IS NOT DESIGNED NOR INTENDED FOR USE ON HUMAN SUBJECTS Publication 5720 001 REV B 4 Oocyte Clamp Amplifier Model OC 725C Warner Instruments Control Description The OC 725C is comprised of three functional channels A high impedance voltage sensing channel with capacity compensation and input offset to measure membrane potential a current sensing channel with bath clamp to clamp the bath and measure the membrane current and a high voltage amplifier to deliver the clamping current The complete voltage clamp system consists of the OC 725C the voltage recording probe with e
33. o the high side current measuring mode by setting a dip switch on the main cir cuit board 1 First disconnect the power cord from the wall 2 Remove the two screws at the rear of the top cover and it off 3 Locate dip switch S10 on the circuit board For normal operation S4 is oz and all other 7 switches are off 4 To convert to high side current measuring mode turn 4 off and turn S2 5 and S7 on All other switches should remain in the off position Optional voltage headstage The optional DIFFERENTIAL INPUT VOLTAGE RECORDING HEADSTAGE Model 7255DD is used in applications where the bath clamp headstage cannot be effectively employed Two examples are 1 situations where the solution path from oocyte to ground is very long and 2 when recording from two oocytes in a common bath Two 1 mm input jacks are located on the side of the headstage v DIFF and GND A shorting jumper is supplied and is used for normal single ended recording For double ended recording the jumper is removed and a v DIFF ELECTRODE is connected to the v DIFF jack as shown below If a shield between the voltage and clamp current electrodes is used it should be connected to the headstage ground Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Comments and Recommendations Membrane damage Recording from the same cell at a later time requires that the cell remain healthy during the interim in
34. onitored in the x10 range Large currents above 100 uA require the x0 7 range Since there is overlap in the ranges the current being measured may be monitored in one of two ranges in which case the choice may be made on the basis of noise or clamp speed Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Setup Cont d Gain select Headstage resistor In output range V pA In max output MA Maximum meter reading x0 1 10 kQ 0 01 1 0 10 1000 199 9 pA x1 0 100 kQ 0 1 10 1 100 19 99 pA x10 1 MQ 1 0 100 0 1 10 1 999 pA Other gain range selection considerations The intrinsic noise of the current measuring circuit is a function of the bath clamp feedback resistor with a larger resistor offering lower noise and greater signal resolution For comparisons the following measurements were made with a shorted input with the standard 0 5 uF capacitance model cell and with a 0 22 uF capacitance modified model cell All readings were recorded at 1 kHz 8 pole Bessel and are RMS Feedback Resistor Noise Shorted Input Standard Model Cell 0 5 pF Modified Model Cell 0 22 pF 10 KQ 75 pA 6 0 nA 4 4 nA 100 KQ 28 pA 5 5 nA 4 4 nA 1 MQ 22 pA 5 0 nA 4 0 nA A lower value feedback resistor increases the speed of the bath clamp and can hand
35. ould be placed as close to the oocyte as sense possible since this point is the virtual ground node and on the same side as the voltage recording electrode The I electrode or the agar bridge associated with it on the other hand can be placed at a greater distance from the oocyte and should be on the same side as the current electrode It is recommended that the user not directly expose the electrode wires to the perfusate if the recording session is to last for more than a few minutes Instead agar bridges should be employed to provide a circuit between these electrodes and the bath This protects the cell membrane from the potential adverse effects of the silver wire Single oocyte setup with indirect ground Single oocyte studies are best accomplished with the setup shown in Figure A This method uses the bath clamp headstage to establish the bath ground and is preferred for two reasons 1 current readings with the bath clamp will have the lowest noise level and 2 proper ly placed bath clamp electrodes will negate the need for series resist ance compensation CURRENT ELECTRODE KOLDER AND CABLE 2 BATH RESISTANCE a BATH REFERENCE ELECTROOCE Single oocyte setup with direct ground Applications where use of the bath clamp is not suitable such as those with a very long solution path to ground can be configured using the alternate method of directly grounding the bath as shown in Figure B In this configuratio
36. rner Instruments Control Description Conto potential is displayed by the meter or reported at the V x10 output BNC at 100 mV MQ The V BUZZ push button facilitates penetration of the voltage elec trode by producing a 1 KHz square wave at the pipette tip The V x10 ourPUT BNC reports the membrane voltage in mV multi plied by 10 NEGATIVE CAPACITY COMPENSATION C has been added to the OC 725C allowing for its use as an electrometer in intracellular measurements Input capacitance up to 45 pF can be neutralized using the two asso ciated controls A lit LED indicates the active status of this circuit In general negative capacity compensation is not useful for oocyte clamp applications since clamp speed is a function of 1 the current electrode resistance 2 the RC time constant of the oocyte typically 1 MQ in parallel with 0 5 uF and 3 the compliance voltage of the clamp current Bath electrodes The BATH ELECTRODES control block contains the BATH PROBE connec tor the CURRENT METER the GAIN SELECT switches and the I MONITOR out impr Vo mv put BNC s BATH PROBE The CURRENT METER reads the voltage V of the current electrode when the CLAMP MODE SELECTOR switch is off see CLAMP section A lit LED indicates voltage readings in mV When in clamp mode CLAMP MODE SELECTOR Switch set to slow or fast the CURRENT METER displays the current d sensed by the bath electrode A lit LED indica
37. tes current readings in uA Instrument gain is set by the two GAIN SELECT controls Gain is select ed by a 7 position GAIN SELECTOR switch which ranges from 0 1 to 10 in 1 2 5 steps and a 3 position toggle switch which selects the gain multiplier x0 1 x1 0 and x10 LED s indicate the gain multiplier selection The combination of these controls allows gain to be set from 0 01 to 100 Publication 5720 001 REV B Oocyte Clamp Amplifier Model OC 725C Warner Instruments Control Description Conto Current outputs are available from the 1 MONITOR BNC at full band width 10 kHz and from the I MONITOR FILTERED BNC which is fil tered at 1 kHz by an integral 4 pole Bessel filter Clamp section The crame control block contains the MODE SELECTOR switch as well as the GAIN and DC GAIN controls The CLAMP MODE SELECTOR switch selects for slow and fast clamp speeds or for off These choices are described below off In the off position the clamp amplifier is disconnected from the current electrode The voltage difference between the current elec trode and the bath electrode V mV is read on the METER in the BATH ELECTRODES control block This information is also available at the V X10 output BNC on the rear of the instrument NOTE All cLAMP controls are disabled by turning the CLAMP MODE SELECTOR Switch to the off position Be sure to switch this control to the off position before handling electrodes
38. th the associated toggle switch COMMAND IN 10 input BNC Command signals from an external generator or computer con nected to this input are attenuated by 10 Maximum input is 10 V Current electrode The CURRENT ELECTRODE block includes CURRENT ELECTRODE Vo the V OFFSET ELECTRODE TEST and V BUZZ heec controls This section also contains the T e OVER VOLTAGE indicator and a DIN con erint nector for the current electrode V OFFSET With a range of 200 mV center zero this control is used to OVER VOLTAGE EJ ELECTRODE Ve BUZZ adjust the offset voltage of the current electrode Use this control to establish a zero reference before impaling the oocyte Once the oocyte has been pierced the resting potential can be read from V x10 output BNC or on the CURRENT METER BATH ELEC TRODES Section ELECTRODE TEST voltage proportional to the resistance of the current electrode 10 mV MQ will be displayed on the meter by depressing the ELECTRODE TEST push button when the CLAMP MODE SELECTOR switch is in the off position V BUZZ The V BUZZ pushbutton facilitates penetration of the voltage electrode by producing a 1 kHz square wave at the pipette tip OVER VOLTAGE LED If the voltage at the current electrode exceeds 160 V the OVER VOLTAGE lamp will light An alarm will also sound when the rear panel ALARM switch is in the on position Publication 5720 001 REV B Oocyte Clamp Amplifier Mode
39. trophysiological configuration is more forgiving than the patch clamp it is still important to minimize mechanical motion The platform for your experimental setup therefore should be mechani cally well isolated This will reduce leakage around the electrodes making the clamp more effective and reducing noise in your recordings The latter is especially important when recording responses of certain ligand gated channels where membrane potential changes may only be a few mV Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Comments amp Recommendations Contd In addition to mechanical isolation the setup must also be isolated from external electrical noise sources These include motors lamps and wiring The platform should be shielded from these sources of electrostatic radia tion with a Faraday cage All equipment within the Faraday cage should be grounded to the rear panel instrument circuit ground and is best achieved by connecting everything including the cage to a ground bus within the cage Then only one wire is run from the setup to the instrument ground You will need to mount the voltage recording electrode headstage and the current injecting electrode on micro manipulators They need not be hydraulically driven but such drives will minimize the damage to the cell during and after penetration and will make for better seals around the elec trodes Another advantage gained by reducin
40. ublication 5720 001 REV B J Oocyte Clamp Amplifier Model OC 725C Warner Instruments A Procedure for Recording from Oocytes 12 The potential across the membrane will now be displayed on the VOLTAGE ELECTRODE METER 13 Now advance the current electrode until its tip is slightly depressing the plasma membrane of the cell and depress the V Buzz pushbut ton Similar to the voltage electrode BUZZ the current electrode BUZZ produces a 1 V 1 kHz oscillation across the current electrode This disrupts the cell membrane and causes the tip of the electrode to impale the cell with no further movement of the micro manipulator Once again if the Buzz technique fails to cause penetration further advance the current electrode until it pops through the membrane Clamping the cell 14 Activate the clamp b 15 Adjust CLAMP GAIN control clockwise as far as possible without illumi nating the OVER VOLTAGE LED located in the CURRENT ELECTRODE section NOTE If the ALARM signal switch on the back panel is oz an alarm will sound when the OVER VOLTAGE LED illuminates 16 The clamped membrane potential can now be observed over time or it can be manipulated by selecting the desired polarity and amplitude with the controls located in the COMMANDS section Alternatively you can control the COMMAND voltage externally from a computer by leav ing the POLARITY toggle switch in the off position and connecting the appropriate analog
41. under nearly any condition The AC clamp gain is variable up to 2000 An additional DC gain of 10 may be employed for high conductance cells or leaky oocytes Two clamp speeds are available The Slow mode is used for screening oocytes or for applications not requiring rapid response times The Fast mode is used for accurate voltage clamping of fast whole cell currents Response time in the Fast mode is 350 us 10 9096 rise time when applying a 100 mV step to a model cell Bath Clamp Headstage The current measuring range of the OC 725C bath clamp headstage is extended at both ends by a 3 position range multiplier This allows smaller currents to be amplified to usable levels while larger currents up to 1 mA can be recorded without saturation The unique design of the bath clamp eliminates the need for series resist ance compensation It provides an accurate measurement of bath current by creating a virtual ground in the bath while simultaneously clamping the bath potential at zero Dual Oocyte Studies Studies involving two oocytes in a common bath requires two clamp amplifiers The bath clamp headstage cannot provide effective clamping because it cannot separate the individual currents from the combined current appearing in the bath The OC 725C solves this prob lem by the application of an internal switch permitting measurement of the current in series with the current electrode instead of in the bath Additionally an optional differenti
42. witch to off CAUTION The current clamp is capable of high power output 10 mA 180 V and can cause serious injury if not properly handled Publication 5720 001 REV B E Oocyte Clamp Amplifier Model OC 725C Warner Instruments Using the Model Membrane The model cell supplied with the OC 725C can be used for two purposes First novice users will find it a convenient tool for gaining experience in the operation of the instrument Additionally it is a convenient tool for trou ble shooting since the function of the instrument can be quickly checked A schematic of the model cell is shown below The oocyte is represented by a 1 MO resistor shunted by a 0 47 uF capacitor The voltage and current electrodes are each represented by 1 MQ resistances and the bath probes are represented by the 10 kQ resistors tobath 4 10k gt 1M 1M clamp WaA M to voltage to current probe cable Initial instrument settings Connect the model cell to the OC 725C as shown on its cover Be sure to connect the ground wire to the ground mini jack on the side of the bath probe Connect the Vm x10 BNC and the 1 Moniror BNC on the OC 725C to an oscilloscope Set the instrument panel controls as follows and turn the POWER SWITCH on Control Control block Setting POWER SWITCH off V n OFFSET VOLTAGE Center of rotation ELECTRODE approximately 5 turns OUTPUT GAIN BATH ELECTRODES 0 1 V uA
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