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OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF

1. 0 Electronic Instruments for the Life Sciences GO ade OPERATING INSTRUCTIONS AND SYSTEM DESCRIPTION OF THE TURBO TEC 05X and TEC 10CX TWO ELECTRODE CLAMP SYSTEMS Please see also Additional Information on TEC 05X D oa eme E SEDLE pro CS TURBO TEC 10CX E O E 3 a VERSION 2 8 npi 2013 npi electronic GmbH Bauhofring 16 D 71732 Tamm Germany Phone 49 0 7141 9730230 Fax 49 0 7141 9730240 support npielectronic com http www npielectronic com TABLE of CONTENTS TABLE e D CK RR KE 2 OSAFETY REGULATIONS csiiinsinpici n ai dd ir 4 da AAA O AN 5 About this MIRA zk SV ARANA rn 3 Important Literat re iii a di dai dal tn 3 DVO EVIE alae ccna tah en saad AAA E nn 3 CellWorks EE SYSTEM DESCRIPTO Nini ege 6 2 0 GENERAL DESCRIPTION EE 6 Selection RE 6 ACCESOS kase o i Eege 6 21I POTENTIAL EECH REM NEE 7 Arrangement of the EN 7 Capacity Compensatii iia 7 Offset E 7 Current Electrode Potential Recording 0d as amp Potential Monitor and Audio Monitor atinada a isa ar A AAA A aaa 8 2 2 CURRENT INJECTION AND CURRENTMEASUREMENT noone 8 CUTIE EE 8 Output Current Zero C HEADSTAGE BIAS CURRENTI nn nnonnnnn nen Current Injection Bandwidth italia dee Dee Geet 9 Capacity Compensation current electrode optional 9 EE 9 Current Measurement A EE EE EE a 10 Current Monitor and Current Output Sensitivity oooooocononoccncncnonnnnnnnnnnonnncnnnonnnnnnn nn ncnnnnnnos 10 T
2. should be avoided all ground points should originate from a central point IMPORTANT The system ground can be disconnected or connected to the m ains ground on the back of the instrument 19 4 2 TESTS AND TUNING PROCEDURES General Considerations The amplifier must be in the current clamp mode when first turned on All system s need a warm up period of about 20 30 m inutes The instrum ents should be calibrated and used for measurements only after this time All sym metrical offset adjustm ent have th e zero position at 5 00 on the respective scale Before turning on the instrum ent all offset c ontrols should be set approximately at this position and all other controls and adjustments should be at zero All system s based on feedback circuits such as capacity com pensation controls or voltage clamp gain must be on a low position close to zero when starting the tuning procedure The INTEGRATOR part of the PI controller must be set OFF before switching to VC mode Basic connections The basic connections for testing and using a TEC system s are given in figs 4 6 The minimum equipment needed is a stim ulus unit and an oscilloscope preferable digital storage scope Usually a com puter based data acquisiti on system is used for experim ents This is connected in a similar manor i e ADC anal og to digital converter to the outputs and DAC digital to analog converter to the inputs of the TEC system For a detailed descript
3. 94us and T 168115 Maximum speed of response The speed of an ideal VC system is lim ited only by the m aximum current delivered by the current source dUm dt max Umax Cm Roi dUm dt max 150V 0 1 uF 1 MQ 1500 V s 1 5 mV us To reach 150 mV would last 100 us provided that the clamp has an ideal characteristic Now we can calculate the m inimum bandwidth of a real clam p system necessary for ideal behavior T 8 4 Te 100 us gives Te 12 us BW 1 2n T 13 kHz If we assume that Te is determ ined by 70 80 by the tim e constant of the current electrode i e Ta 10us if T o 12us it is clear that with electrode resistances in the range of 500 k the total capacity related to the current inj ecting electrode can be maximum 20pF Maximum cable length in this case is 15 20 cm A cable of 0 5 1 5 m has a capacity in the range of 50 200 pF W ith such a capacity and an electrode resistance of 1 MQ T is in the range of 50 200 us and the speed of response would be in a range of 0 5 2 ms Conclusions For adequate VC experim ents a clam p gain of 1 5 m A V i e 100 500 internal gain with a current source calibration of 10 A V is nece ssary Therefore with pulse am plitudes of 100 200 mV the operational am plifiers in the gain stages will be saturated causing nonlinear components in the capacitive transients The maximum speed of response is determ ined by the cell capacity the m aximum available current an
4. current sensitivity selection amplifier and low pass filter CURRENT TRANSIENT COMPENSATION TEC 10CX only Capacitive transient Al A2 A3 T1 T2 T3 and leakag e current LINEAR com pensation in voltage clam p mode BYPASS ON switch If set to BYPASS the current transient compensation unit is switched off i e am plitude and tim e constant settings ofthe transientcom pensation unit are not working If set to ON the current transien t com pensation unit is active The LINEAR component is not affected by this switch Caution In current clam p m ode the al a3 and LINEAR controls m ust be in the zero position BATH POTENTIAL mV TEC 05X only signal at the REF electrode CURRENT FILTER Hz Current output low pass filter CURRENT OUTPUT SENSITIVITY Am plification switch f or the CURRENT OUTPUT signal 0 1V LA 10 V LA in seven steps HOLDING CURRENT 0 switch Holding current control current clamp mode CURRENT STIMULUS INPUT Current stimulus input in current clamp mode MODE OF OPERATION TEC 10CX Control unit for selection of the operation mode Rce Resistance test of current electrode CC Current clamp mode VC Voltage clamp mode Rpa Resistance test of potential electrode MODE OF OPERATION TEC 05X Control unit for selection of the operation mode BRIDGE Bridge mode for potential electrode CC Current clamp mode OFF In this position the amplifier does not apply any voltage or current to the cell
5. time constant and many small time constants These small time constants can be added to an equivalent time constant Te In case of the TEC control chai n the large tim e constant is form ed by the cell m embrane several hundred ofm s and the sum of sm all tim e constants results from the microelectrodes and the electronics a few te n us Here we consider only the proportional part of the PI controller We also do not consider possible improvement of clamp performance due to series resistance compensation 6 8 and 20 for details General Considerations For the TEC system s the sm all time constants are at least two orders of m agnitude below the large time constant The large tim e constant is the tim e constant of the m embrane and the equivalent tim e constant is composed of the time constants of the electrodes amplifiers etc 25 Ta SBA i ar The performance of a clamp system can be improved if a voltage controlled current source is used for the current injecting electrode In this case the very large tim e constant hundreds of milliseconds formed by the electrode resistance and the cell capacity can be ignored since the output of the clam p circuit is a current whic h flows regardless of the resistance of the injecting microelectrode see reference 20 for de tails Thus the perform ance of the clam p is no longer depending on the electrode resistan ce as long as the current source is not saturated The
6. C W 20 40 60 80 100 120 140 160 time ms Cstray and Ve compensated potential mV current nA 60 3 0 tem 50 25 40 2 0 30 Tom 1 5 20 10 10 Go 0 DD 0 20 40 60 80 100 120 140 160 time ms potential current Figure 3 Artifact caused by the recording electrode The measurements were done using a cell model with 100 MQ membrane resistance 100 pF membrane capacitance and 100 MQ electrode resistance A Cstray and Nep not compensated bridge not balanced B Cotray compensated and Ver not compensated C Cotray and Nee compensated bridge balanced Cm membrane capacitance Cstray electrode stray capacitance Ro electrode resistance Rm membrane resistance Tcm time constant of the cell membrane Vert potential drop at Ro Sample Experiment If you intend to do first single electrode ex periments and then experim ents with two electrodes impaled follow the guideline below If you plan to use only the POTENTI AL electrode for record ing the m embrane poten tial or operating in BRIDGE mode do all adjustments only for the POTENTIAL electrode and skip the CURRENT electrode Note W e recomm end to disconnect the CURRE NT headstag e if only single elec trode operation in BRIDGE mode is used The am plifier work s fine even ifno head stage is connected to the CURRENT HEADSTAGE connector DO LU UL L UL a Single electrode operation If not already done compensate the BIAS CURRENT of both electrode
7. SS OSC SHUT OFF POTENTIAL RECORDING POT MONITOR Rs COMP 4 LIMITER Be Lo IA 7 Ge u A A INTEGRATOR 7 FAST 1 C COMP SALN gw En SE rr gt ERRUR E REF 8 7 MEDIUM BUFFER PI CONTROLLER gt RISE TIME 1 K HOLDING POT KEE 7 SLOW STEP H ES S V COMMAND INPU CUR TRANSIENT COMPENSA gt E MA UNCOMPENSATED CURRENT MONITOR COMPENSATED A GAIN 7 FILTER ON FIG 1 Equivalent Circuit Diagram of TEC 10 Voltage Clamp System POTENTIAL QUTPYT Fig 2A EQUIVALENT CIRCUIT OF TEC AMPLIFIER CELL DEE Fig 2B BLOCK DIAGRAM OF VC MODE INTEGR TOR OSCILLATION SHUT OFF POTENTIAL RESISTANCE CURRENT OUTPUT SENSITIVITY
8. are in the MQ range it is necessary to use a high voltage current source 150 for current in jection The TEC standard version has an output compliance of 150 V i e the maximum current is 150 uA 1 MQ The current range of the various TEC versions is determined as follows TEC 05X 150nA 100 MO or 1 5 LA 10M for large cells e g invertebrate neurons TEC 10CX 150 A 1 MA for very large cells e g oocytes Some current headstages are equipped witha switch for the selection of different current ranges see options below For the standard 150 V headstage the ranges are Option 1 x0 1 x1 x2 x5 Option 2 x0 1 x0 2 x0 5 x1 x0 1 range 15 pA 10 MQ x0 1 range 15 pA 10MQ xl range 1501A 1 MQ x0 2 range 30 LA 5 MQ X2 range 300 LA 500 KQ x0 5 range 75 pA 2 MQ x5 range 500 1A 200 KQ xl range 1501A 1 MQ WARNING Always adhere to the appropriate sa fety m easures see Safety Regulations Introduction and Installation chapters when usi ng these instrum ents In particular always shut power off when changi ng or adjusting electrodes Always turn pow er off w hen connecting or disconnecting headstages or other components from the 19 cabinet Current Measurement The use of the current source output allows that the current is m easured en route to the electrode an im provement in accuracy on th e virtual ground m ethod which requires an additional headstage The current source m ethod also provides anim proved
9. article by St hmer et al Methods in Enzymology Vol 207 In addition a unique oscillation shutoff circuit prevents the cell from damage if oscillations occur Since the voltage and current clam p techniques are standard techniques of electrophysiology for a review see Methods in Enzym ology Vol 207 Sm ith et al 1985 or Standen et al 1987 Kettenm ann amp Grantyn 1992 Ogden 1994 only a short procedural description follows based on the diagram s of Fig 1 and Fi g 2 Terms and abbreviations in capital letters in the text correspond with the labels on the front panel Selection and Options The following versions are available TEC 03X standard oocyte amplifier separate manual available TEC 03X CW special version for CellWorks software TEC 05X large cells muscle invertebrate cells with fine tipped electrodes please contact npi electronic for details TEC 05CX CW special version for CellWorks software TEC 10X TEC 10 with digital control of cu rrent filter and gain without transient compensation unit TEC 10X CW special version for CellWorks software TEC 10CX TEC 10 with digital control of current filter and gain TEC 10CX CW special version for CellWorks software Accessories TEC system s are delivered with two headstag e power chord m anual and a set of cables connectors for the reference ground and the curre nt electrode connector Special headstages microelectrode holders and cell m odels TEC Cel
10. eability to Gating Charge of rBIIA Sodium Channels and Sodium Influx in Xenopus Oocytes Biophys Journal Vol 79 2434 59 Babini E Paukert M Geisler H S z Griinder S 2002 Alternative Splicing and Interaction with Di and Polyvalent Cations Control the Dynam ic Range of Acid sensing Ion Channel 1 ASIC1 J Biol Chem 277 41597 41603 Jenke M Sanchez A Monje F Stuhm er W Weseloh R M amp Pardo L A 2003 C terminal dom ains im plicated in the functi onal surface expression of potassium channels EMBO J 22 395 403 Paukert M Hidayat S amp Grunder S 2002 The P2X 7 receptor from Xenopus laevis formation of a large pore in Xenopus oocytes FEBS Lett 513 253 258 Schmitt B M and H Koepsell 2002 An Improved Method For Real Tim e Monitoring of Membrane Capacitance in Xenopus laevis Oocytes Biophys J 82 1345 1357 Nagel G Ollig D Fuhrmann M Kateriya S Musti A M Bamberg E amp Hegemann P 2002 Channelrhodopsin 1 a light gated proton channel in green algae Science 296 2395 2398 Est vez R Schroeder B C Accardi A Jentsch T J amp Pusch M 2003 Conservation of Chloride Channel Structure Revealed by an Inhibitor Binding Site in CIC 1 Neuron 38 47 59 Rettinger J amp Schm alzing G 2003 Activa tion and desensitization of the recom binant P2X1 receptor at nanomolar ATP concentrations J Gen Physiol 121 451 461 Verri T Kott
11. frequency response of the voltage clamp control circuit Current Monitor and Current Output Sensitivity The TEC systems have two current outputs CURRENT OUTPUT UNCOMPENSATED the current signal directly obtained from theh eadstage 0 1 V pA i e 1 V at this BNC corresponds to a current of 10 A injected into the cell standard version The current from the headstage is also displayed on the digital di splay lower display This signal is am plified and filtered for better presentation giving the CURRENT OUTPUT signal The am plification is perform ed by an am plifier with seven gain f actors f rom 0 1V pA 10V uA which corresponds to an amplification of x1 x2 x5 x10 x20 x50 x100 The selection can be set by a rotary sw itch CURRENT OUTPUT SENSITIVITY The following calibrations for the signal at the CURRENT OUTPUT BNC result TEC 05X 0 1 0 2 0 5 1 2 5 and 10 V nA display XX XX nA TEC 10CX 0 1 0 2 0 5 1 2 5 and 10 V A display XX XX LA The position of the CURRENT OUTPUT SENS ITIVITY switch is m onitored by a DC voltage 1 V switch position 1V 7V av ailable at the rear panel MONITORING OUTPUTS CURRENT OUTPUT SENSITIVITY BNC connector Example A current sensitivity of 1 V uA at CURRENT OUTPUT BNC corresponds to a voltage of 4 V at the CURRENT OUTPUT SENSITIVITY BNC connector Transient Compensation TEC 10CX The TURBO TEC 1 0CX series provides a com pensation unit for the suppression of the capaci
12. if the output of the clamp system is a current source rather than a voltage source in this case the clamp transfer function has the m agnitude of a conductance A V Other advantages of this arrangement are that the clam p current can be determined by a dif ferential am plifier no virtual ground is needed see 6 13 and that th e bandwidth of the feedback system can be altered easily e g for noise suppression during simultaneous patch clamp recordings see 19 21 This output circuit m ust be equipped with large bandwidth high voltage operational amplifiers To avoid deterioration of clam p perform ance caused by electrode overload the output current has to be lim ited by an electronic circuit to a safe level With electrodes in the range of one M Q and a voltage of 150 V the m aximum current will be 150 LA W ith this current a cell with a capacity of 0 1 uF can be depolarized by 100 m V in approxim ately 100 us which com es close to the theoretically possible speed of response without any detectable deviations from the command level With an output compliance of 225 V and a x2 or x5 range current injecting headstage currents up to 500 uA can be injected see 6 and 15 The speed of response and the accuracy of a two electrode clamp system is determined by the cell capacity the resistance of the current in jecting m icroelectrode which lim its the maximum amount of injected current and the e quivalent time constant and accuracy of the po
13. instrum ent provides a bias current adjustment with a 10 turn potentio meter ca 0 5 of the current range The tuning procedure is described in the INSTALLATION chapter Current Injection Bandwidth On all TEC system s for oocyte recordings the ba ndwidth of the current injection electronics can be lim ited to approxim ately 10 Hz by m eans of a switch BANDW on the current headstage see Fig 5 This allows the use of a patch clam p amplifier for the recording of channel currents sim ultaneously to m acro currents recorded with the TEC system without excessive noise from the two electrode clamp loop see 2 5 Low Noise Mode WARNING If the bandwidth of the current headstage is set to 10 Hz som e functions such as Rce current electrode resistance test do not work properly Capacity Compensation current electrode optional The TEC 05X amplifier are equipped with a cap acity compensation for the current electrode CUR EL C COMP 10 turn potentiom eter This increases the speed of the voltage clam p control circuit when using high resistance gt 1 MQ microelectrodes WARNING Capacity com pensation is based on positive f eedback Theref ore overcompensation causes oscillations ringing wh ich can deteriorate the preparation or the recording electrodes Therefore the control m ust be handled with care and before im paling a new cell must be set to 0 Current Range Since the resistances of the microelectrodes
14. the m ode of operation The active mode is indicated by LED s The switch labeled MODE OF OPERATION located below the displays is used to select DHC VC CC OFF BRIDGE or EXTERN mode DHC and BRIDGE mode are optional In the EXTERN mode the mode of operation can be determined by a TTL pulse applied at the MODE SELECT INPUT BNC connector LO CC HI VC If connected to CellWorks the system can be controlled from software 13 3 CONTROLS and CONNECTORS 3 1 FRONT PANEL A general view of the TEC f ront panels is given in Fig 3 The users elem ents will be described starting from bottom to top from the lower right to left The current clam p controls and those for adjustment of the current signal are oriented on the right side of the front panel The keys for the digital control units and the digital display are found in the m iddle and the elements for the voltage clamp mode are oriented on the left side of the panel HEADSTAGE INPUTS Plugs for connection of the headstages POTENTIAL ELECTRODE C COMP Capacity ne utralization potential electrode current electrode optional POTENTIAL CURRENT ELECTRODE OFFSET Offs et compensation potential electrode current electrode CURRENT HEADSTAGE BIAS Zero setting for the current source current electrode CURRENT OUTPUT FROM HEADSTAGE Current signal from the current headstage 0 1V nA CURRENT OUTPUT FILTERED Current output signal passed through transient compensation TEC 10CX only
15. the potential headstage the user can penetrate a cell m easure membrane potential and apply current pulses in CC mode In BRIDGE mode see MODE OF OPERATION switch the TEC 05X operates like a single electrode bridge amplifier see also separate manual 3 4 CURRENT HEADSTAGE WARNING LETHAL HIGH VOLTAGE CURR EL Connection for the current electrode GND Ground connector RANGE x1 x0 1 or x0 1 x1 x2 x5 or x0 1 x0 2 x0 5 x1 Selection of the current range option BANDWIDTH wb 10 Hz Selection of the bandwidth see 2 5 Low Noise Mode WARNING If the bandwidth of the current headstage is set to 10 Hz som e functions such as Rce current electrode resistance test do not work properly 18 4 INSTALLATION 4 1 GENERAL CONSIDERATIONS Safety IMPORTANT Please f ollow strictly all regulations outlined in chapter 0 SAFETY REGULATIONS In working with the TURBO TEC systems always adhere to the appropriate safety m easures for handling electronic devices This instrum ent functions with a high voltage outlet CUR EL plug on the current headstage After turning on this instrum ent it must be ensured that the interior contact of this plug cannot be touc hed In addition it is extrem ely important that the instrument is turned off when changing or adjusting either electrode In addition both headstages contain very se nsitive FET am plifiers which can be dam aged with electrostatic charge and must therefore b
16. 3 ELECTRODE RESISTANCE MEASUREMENT Resistance measurement modes for both m icroelectrodes R ra and R ce are included in this device in order to test the function of the electrodes These test units operate independently of any other adjustm ents This is possible under the condition that all m icroelectrodes are in contact with a grounded bath zero potential The m easured resistance is independent of tip potentials and is automatically displayed on the digital display in MQ The m easurement is perform ed by applying square current pulses of a few nA to the respective microelectrode The voltage deflecti on caused by this injection is recorded and processed to give a direct reading in M Q on the digital display The electrode resistance test is also a test of the correct function of the respective headstage WARNING RESISTANCE TEST m ode gives only a correct value with high resistance electrodes if the capacity is com pensated correctly Furthermore with a headstage with four ranges see above the RESISTANCE TEST has to be carried out in x1 position Only in x1 position the value is displayed correctly 11 2 4 CURRENT CLAMP MODE CC In the current clam pm ode the cell s reaction to current injections is m easured Current injection is perform ed by m eans of a current source connected to the current injecting microelectrode regardless of the electrode re sistance see Fig 1 Therefore only a current input conditioning unit i
17. Before using any device please read manuals and instructions carefully Always use a three wire line cord and a mains power plug with a protection contact connected to mains ground protective earth Check for appropriate line voltage before connecting any system to mains Before opening the cabinet disconnect mains power plug Disconnect mains power plug when replacing the fuse or changing line voltage Replace fuse only by appropriate specified type STATIC ELECTRICITY Electronic equipment is sensitive to static discharges Some input devices such as headstages are equipped with very sensitive FET am plifiers which can be damaged by electrostatic charge and must therefore be handled with care This can be avoided by touching a grounded m etal surface when cha nging or adjusting the electrodes If a headstage is not used the input should always be connected to ground by using an appropriate connector or with aluminum foil wrapped around the headstage VERY IMPORTANT Always turn power off when conn ecting or disconnecting headstages or other components from the 19 cabinet CURRENT INJECTION HIGH VOLTAGE HEADSTAGE The current injection headstage has a 45 V or 150 V output compliance After turning on the instrument it must be ensured that the interior contact and the shield of the electrode plug and of the cable which is connected to this plug cannot be touched VERY IMPORTANT Always turn power off when changing or adjusting the e
18. CTRODE control unit The operation is analog to the controls for the potential electrode The CURRENT ELECTRODE co ntrol unit consist of HEADSTAGE BIAS potentiometer OFFSET potentiom eter and CAPACITY COMPENSATION potentiometer HEADSTAGE BIAS Potentiometer to compensate for the HEADSTAGE BIAS current of the current electrode ten turn potentiometer clockwise see also Adjustment of the CURRENT HEADSTAGE BIAS CURRENT control in the user manual page 21 ff OFFSET Potentiometer to com pensate f or th e OFFSET potential of the cu rrent electrode ten turn potentiometer Note position 5 corresponds to 0 mV OFFSET Fine CAPACITY COMPENSATION Control for the com pensation of the cap acitance of the currente lectrode ten turn potentiometer clockwise Important Capacity co mpensation of both elec trodes is sp lit into two controls Coarse controls at the headstages and a fine controls at the front panel of the amplifier The coarse controls are used to set a range for the fine controls Especially in V C mode the coarse controls are very sensitive and can cause oscillations if overcompensated Note The electrode resistance is displayed correctly only if the capacity for the respective electrode is compensated properly PENETRATION ELECTRODE CLEAR control unit BUZZ push button H Red push button to activate the BUZZ ELECTRODE CLEAR unit Note The electrode for buzzing is selected with the switch for the POTENTIA
19. DE ELECTRODE BATH GROUND REFERENCE ELECTRODE 1300N 1139 19d y 3P0 112913 1U944N 3P0112913 TP91T1U310d Electronic E Instruments for the Life Sciences Additional Information on TEC 05X with BRIDGE Mode BUZZ Function and SEC Headstages TEC 05X npl 9 9 9 9 80 0 QO The TEC 05X comes with two SEC headstages one connected to CURRENT HEADSTAGE the other connected to PO TENTIAL HEADSTAGE maximum current 120 nA into 100 MQ With this amplif ier itis possib le to perf orm current clamp experiments with only one electrode impaled as well as s tandard two el ectrode current CC an d voltage clam p VC experiments Single electrode m easurements in BRIDGE mode are d one with th e electrode connected to the POTE NTIAL HE ADSTAGE which has the ability to m easure potentials like th e s tandard po tential head stage and to inject cur rent s imultaneously in BRIDGE mode maximum current 120 nA Additional Front Panel Elements BRIDGE BALANCE MQ potentiometer and range switch If current is passed through the r ecording electrode the potential deflection caused at the electrode resistance is compensated with this control ten turn poten tiometer clockwise ca librated in M A switch 1s used to set the el ectrode resistance range 1000 MQ maximum 100 MQ TURN or 100 MQ maximum 10 MQ TURN i e 80 0 on the dial corresponds to 80 MQ if the switch is set to 10 MQ TURN CURRENT ELE
20. In this systems the capacity compensation of both electrodes 1s split into two controls for each electrode the coarse con trol in the headstage and a the fine contro at the front panel of the amplifier The aim of the f irst part of the tu ning procedure is to se tthe coarse capacity compensation at the headstag e sothat an optim al wide range of CAPACITY COMPENSATION control at the amplifier is achieved Note If the cell model is used the switch should be in position BATH in order to simulate the electrodes in the bath Otherwise the capacity of the cell model is always present leading to a slurred signal The tuning is analogue for both electrodes and described only for the potential electrode Both electrodes should be in the bath preferably as deep as they will be during the experiment For the CURRENT ELECTRODE watch the signal at Cer O Set the CAPACITY COMPENSATION control of the POTENTIAL ELECTRODE at the amplifier to a value around 3 and turn COARSE CAPACITY COMPENSATION at the potential headstage to the leftmost position Set the amplifier to OFF mode Push the ELECTRODE RESISTANCE s witch to POTENTIAL and wa tch the POTENTIAL OUTPUT P rr The signal at POTENTIAL OUTPUT P gr should be tuned with the COARSE CAP ACITY COMPENSATION at the p otential headstage to be come as square as possible LU Second part fine tuning Now the basic setting of the CAPACITY COMP ENSATION is achieved Since the electrode parameters change d
21. L DISPLAY BUZZ REMOTE connector BNC connector to attach a rem ote switch to the BUZZ unit Either a hand held switch or a foot switch can be used to operate the BUZZ circuit rem otely The remote device is connected via a grounded BNC cable BUZZ DURATION potentiometer Potentiometer to set the duration of the BUZZ approx 5 to 100 ms Mode switch Switch for selection of the BUZZ ELECTRODE CLEAR mode Imax BUZZ using the maximum positive current Imax BUZZ using the maximum negative current BUZZ BUZZ using oscillations cause d by overcom pensating the capacity compensation system The overcompensation of capacity compensation yields to very powerful high frequency oscillations OFF BUZZ function is disabled POTENTIAL ELECTRODE BIAS CURRENT control c Control for the compensation of the BIAS current of the potential electrode Note Because the potential electrode in this amplifier is able to work in BRIDGE mode 1 e to inject current in to the cell it has like the CURRENT ELECTRODE a control for cancellation of the BIAS curren t This control works analo gue to th e control of th e current electrode s ee also Adjustment of the CURRENT HEADST AGE BIAS CURRE NT control in the user manual page 21 ff Fine CAPACITY COMPENSATION A Control for the com pensation of the capacita nce of the pot ential electrode ten turn LM potentiometer clockwise Important Capacity co mpensation of both electrod es
22. T m 22 Oscillation E 22 5 CONTROL THEORY APPENDIX sssesssccesccescoessccssccesccescocesocssoccssccesccescosesoeessecesccesosesse 23 5 1 THEORY OF OPERATION OF THE TEC SERIES AMPLIFIERS occcoocccconcncnonnnnonnnnnnnncconnanonnnonos 23 EMPIRICAL TUNING PROCEDURE FOR PI CONTROLLERS sccsssseceeseeceeeeeceeececeeeeceeeeeesaeees 25 5 3 SPEED OF RESPONSE AND LINEARITY OF THE CAPACITIVE IRANSIENTS 25 6 REFERENCES E v r 28 7 TURBO TEC SERIES SPECTFICATITONG scsssscsssssssssssssssscssscsssesssssesscsssscsssassssnsses 31 0 SAFETY REGULATIONS VERY IMPORTANT Instruments and components supplied by npi electronic are NOT intended for clinical use or medical purposes e g for diagnosis or treatment of humans or for any other life supporting syst em npi electronic expressively disclaims any w arranties for such purpose Equipmen t supplied by npi electronic shall be operated only by selected trained and ade quately instructed personnel For details please consult the GENERAL TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic D 71732 Tamm Germany GENERAL This system is designed for use in scie ntific laboratories and should be operated by trained staff only General sa fety regulations for operating electrical devices are to be considered AC MAINS CONNECTION In working with instrum ents and components supplied by opt electronic always adhere to the appropriate safety measures for handling electronic devices
23. VC Voltage clamp mode EXTERN if this position is selected the mode of operation can be set by a TTL pulse applied to the MODE SELECT INPUT TTL BNC LO CC HI VC or DHC if installed DHC Dynamic Hybrid Clamp mode option 16 DIGITAL DISPLAYS 3 digits POTENTIAL RESISTANCE membrane potential in m V resistance in M with LED s for the selected unit mV MQ CURRENT current in pA POTENTIAL Switch for upper digital display Electrode switch POTENTIAL ELECTRODE Membrane potential or resistance of the potential electrode CURRENT ELECTRODE Potential of the current electrode or resistance of the current electrode OSCILLATION SHUT OFF Protection against oscillation of the amplifier THRESHOLD Adjustment of the threshold DISABLED RESET Choice of operation DISABLED unit is not active LED red amplifier shut off green turned on in operation HOLDING POTENTIAL mV 0 switch Holding potential control in VC mode VOLTAGE COMMAND INPUT Command signal in voltage clamp mode 10 mV or 40 mV POTENTIAL OUTPUT Per x10 mV or x40 mV Membrane potential output POTENTIAL OUTPUT Cr x10 mV Potential output of current electrode COMMAND FILTER TIME CONSTANT Tim e cons tant of the filter for the VOLTAGE COMMAND INPUT 10 us to 1 ms INTEGRATOR TIME CONSTANT ON FF switch Ti me constant of the integrator of the proportional integral controller PI controller VC OUTPUT LIMITER 0 100 Limits max
24. VipA CURRENT TRANSIENT COMPENSATION mi 1 POWER PL CONTOLLER of D Rca VC BABA VOLTAGE A P MODE OF OPERATION CURRENT nm tea TURBO TEC 10CX Ge r o o e I CURRE Fig 3 TEC 10CX Front Panel FIG 4 CONNECTIONS POWER OSCILLATION PI CONTROLLER POTENTIAL ELECTRODE RESISTANCE CURRENT 5 SHUTOFF POTENTIAL POTENTIAL O ELECTRODE ELECTRODE C HEADSTAGE BIAS CURRENT w d 0 050 m fe ELECTRODE ELECTRODE TURBO TE C z 0 3 DISABLED NORMAL Gain only xa a npi y reese 1k 1 3K 2k 0 5 1 700 a 02 1 1 2 MODE OF OPERATION 500 es Se v e vc CC EXTERN 300 a CURRENT ELE OFFSET 200 10k ao ws PO TENTIAL 100 13k TA aor 20k VILA OFFSET Ge SE CURRENT OUTPUT SENSITIVITY SI CURRENT FILTER mm yu Y A AN SE A A 1V STEP gt CAPACITY O A E O 8V 7V G Mn COMPENSAT Ne g BENE RE E FREQUENCY MONITOR GAIN MONITOR HOLD SC a e VC e SCH e x10 mV x10 mV 10 my gt 14AN p O e O 1V JUA Serie K a a Ai K K GROUND Pel CEL MODE CURRENT CURRENT OUTPUT CURRENT HEADSTAGE POTENTIAL OUTPUT SELECT OUTPUT FROM HEADSTAGE DAC Stimulus generator W AD Conv Oscilloscope FIG 5 HEADSTAGE CONNECTIONS CURRENT HEADSTAGE POTENTIAL HEADSTAGE CAUTION HIGH VOLTAGE 150V ee m E BANDW SC GND SEA ee 10Hz Wb POTENTIAL CURRENT ELECTRO
25. a capacity of ca 10 pF with the stray capacities in the headstage and an electrode resistance of 1 M cell m odel this gives a tim e constant of 10 30115 corner frequencies of 5 15kHz With Cm 0 1uF and Te 20us 8kHz bandwidth the gain can be calculated as LO K 1 25 mA V MO K 2 5 mA V The standard TEC current source has a calibra tion of 10 uA V This means that the gain stages related to the GAIN control on the front panel must provide a gain between 125 250 In the TEC system the gain amplifier is composed of two stages x10 fix and 1 100 variable The maximum gain of the variable gain stage can be set with an internal trim potentiometer If a pulse of 150 m V is applied the output of the first stage is 1 5V while the second stage goes into saturation if the calculated gain values are used Therefore the capacitive transients will have large nonlinear components 26 A response with no saturation effects is obt ainable only with com mand signals below 100 mV If larger m embrane capacities are used in the cell m odel the saturation ef fects start earlier because in this case a higher gain is re quired To improve this behavior system s with higher output com pliance and or headstages w ith x2 x5 or x10 ranges m ust be used which avoid the saturation effect of the gain amplifier see references 6 13 and 14 The speed of response with x1 headstage and 150 V output from the point of view of control theory is T
26. active in Fig 1 and Cell activity in Fig 2 under stable conditions the injected current corresponds directly to the ionic m ovements across the cell m embrane The VC ERROR display shows directly the deviation of the recorded potential from the command signal For an accurate clam p it must be between 0 5 max 1 Current Limit VC OUTPUT LIMIT Under certain experimental conditions it is necessary to limit the current in the voltage clamp mode e g in order to prevent the blocking of the electrode or to protect the preparation This is possible with an electronic limiter which sets the current range between 0 100 Series Resistance Compensation With som e preparations it is not always possible to exclude series resistances despite differential potential recording see Fig 1 These series resistances could cause a current proportional potential error in the voltage clam p mode i e an unwanted change in the membrane potential during a current flow This change can be partially com pensated by current proportional amplification in the control circuit This is done by positive feedback in the control circuit which can very quickly lead to stability problem s Re positioning the electrodes is recom mendable whenever possible use this com pensation procedure only as a last resort Low Noise Mode TEC instruments can be used in a low noise m ode for simultaneous recordings with a patch clamp amplifier For this purpose th
27. as necessary d uring the experiment U Tune the OFFSET to zero and com pensate th e electrode capacitance This is very important since a badly com pensated input capacitance prevents setting the BRIDGE BALANCE to correct values J Determine the resistance of the P OTENTIAL ELECTRODE using the ELECTRODE RESISTANCE switch and set the BRIDGE BALANCE RANGE switch accordingly J Apply current pulses to the electrode at CURRENT STIMULUS INPUT connector J Watch the POTENTIAL OUTPUT P eat the oscilloscop e and adjus tthe BRIDGE BALANCE as shown in Figure 2 using th e BRIDGE B ALANCE potentiometer After adjustment you should see a straight voltage trace without artifacts caused by the potential drop at Re Figure 2 illustrates the BRIDGE BALANCE procedure using a 100M Q resistor that represents the electrode In the upp er diagram the bridge is slightly undercom pensated and in the diagram in the m iddle it is slig htly overcompensated The lower diagram shows a well balanced bridge compensated Important BRIDGE BALANCE and CAP COMP m ust be tuned severa times during an experiment since most parameters change during a recording session Figure 3 shows artifacts caused by uncom pensated stray capacitance and bri dge during recording from a cell It also shows how to cancel these artifacts by tuning with CAP COMP and BRIDGE BALANCE OFFSET deviations can be detected by comparing the readout on the potential display before and after an e
28. cell you should read the mem brane potential at the DISPLAY Set the POTENTIAL DISPLAY switch to POTENTIAL ELECTRODE You are now ready to apply pulses either in tw o el ectrode CC m ode or in BRIDGE m ode In BRIDGE mode curren t stim uli are applied via the potential electrode and in two electrode CC mode stimuli are applied via the current electrode For voltage clamp experiments switch to VC mode Caution The coarse controls at the headstages are very sensitive in VC mode
29. clamp gain in this case has the magnitude of a conductance A V The proportional gain of the clamp system can be calculated as follows references 5 and 12 K C 4T Linear optimum LO aperiodic response no overshoot K C 2T Modulus optimum MO 4 overshoot fastest rise time The gain which should be used ina VC e xperiment is between these two values The overshoot can be reduced by low pass filtering of the command pulse The speed of response of the clamp in case of the modulus optimum can be calculated as T 4 7 Te T 8 4 Te T time until the membrane potential reaches for the first time 100 of the command pulse T tim e to reach steady state within a tolerance of 2 T is roughly the duration of the capacitive transient For a system with dampened overshoot T approaches Ts From these form ulas it is clear that the pe rformance of the clam p is determined by T e Te is determined by the tim e constant of the current injecting electrode 1 e by the electrode resistance stray capacities cable capacities et c Shielded cables have capacities of 60 110 pF m connectors and pipette holders add a few picofarads The potential electrode is equipped with a driven shield and a capacity com pensation circuit theref ore this tim e constant is always much smaller than the time constant associated with the current electrode The time constants of the operational amplifiers can be neglected Example A cable of ca 10 cm has
30. d by means of an integrator for details see refs 5 12 and 13 2 Conseguently amplification of freguencies with a value less than the cut off freguency of the integrator reciprocal to the tim e constant becom es very large which then positively influences the control process The resulting signal is used as the com mand for the current source see Figs 1 and 2 and determ ines the current injection which approaches the set command membrane potential because of the polarity change in the control circuit More details about the functioning of the PI controller are given in the Control Theory Appendix 5 1 The proportional gain is set with the potentiom eter GAIN the time constant of the integrator is set with the poten tiometer INTERGRATOR TIME CONSTANT The integrator can be shut off with a toggle switch In the Control Theory Appendix 5 2 the adjustment of the PI control loop is elaborate d Some considerations concerning the speed of response and linearity are given in Appendix 5 3 VC Error Display The recorded m embrane potential is com pared with the com mand input signal with a differential amplifier giving the VC error signal This signal is applied to the controller where it is am plified and fed back into the current source feedback signal The feedback signal is converted into a current injected through the curre nt electrode into the cell to com pensate the ionic fluxes across the cell m embrane sym bolically called
31. d the command amplitude The real speed of response is determ ined by th e time constant associated with the current injecting electrode It is strongly dependent on the length of the used cable The speed of response and the linearity of the capacitive transients can be improved considerably if a current headstage with a steeper gain x2 20 uA V x5 50 nA V is used especially in combination with a higher output voltage of 225 V TEC 220 System and an improved series resistance compensation see references 2 6 and I3and 15 ST 6 REFERENCES Boulton A A Baker G B and Vanderwol fC H eds 1990 Neurophysiological Technigues Basic Methods and Concepts Humana Press Clifton New Jersey Dietzel I D D Bruns H R Polder and H D Lux 1992 Voltage Clam p Recording in Kettenmann H and R Grantyn eds Practi cal Electrophysiological Methods W iley Liss New York Eisenberg R S and E Engel 1970 The spatial variation of potential near a sm all source of current in a sperical cell J Gen Physiol 55 736 739 Ferreira H G and M W Marshall 1985 The biophysical basis of excitability Cam bridge University Press Cambridge Froehr F 1985 Electronic C ontrol Engineering Made Easy An Introduction for Beginners Siemens AG Berlin amp Munich Greeff N G and H R Polder 1997 An optim ised high current oocyte clam p amplifier with ultralinear low noise response in Elsner N and H W ssle ed
32. d to cell model Rez 1M O Rm 100k Cm 0 1 uF standard headstage lt 80 us with 225 V headstage POWER REQUIREMENTS 115 230 V AC 60 W 1 25 0 63 A fuse SLOW DIMENSIONS 19 rackm ount cabinet 19 483 m m wide 14 355 m m deep 5 25 132 5 m m high weight 11 kg 33 ACCESSORIES PROVIDED Potential headstage standard current headst age other headstages m ay be substituted on reguest with order Cable set and connectors for reference current electrode and ground connectors Power cable Operation manual OPTIONAL ACCESSORIES ordered at additional cost TEC MOD passive model cell ODA active model cell High voltage headstage with four current ranges TEC EH SET electrode holder set TRC 01M TEC 05X system s only Current transient com pensation m odule for TEC 05 amplifiers 34 HU BUFFER S cnn Y gt Cel POTENTIAL Hi OFF SET DIFF CUR REGISTRATION C COMP lt FEEDBACK SIGNAL HU AMP CURRENT INJECTION
33. des hints for the tuning of the voltage clamp control unit and in chapter 6 References a selection of literature is given The last chapter 7 outlines the m ost important technical data of the TURBO TEC amplifiers series Important Literature An excellent introduction to recording techniques preparation of oocytes etc can be found in Methods in Enzymology Vol 207 see ref 21 and the chapter 2 19 by Stuehm er et al in ref 10 The basics of m icroelectrode tec hniques and VC principles are described in a comprehensive m anor in the Plym outh W orkshop Handbook Ogden 1996 see ref 11 Please ref er to chapter 6 REFERENCES f or a more detailed list of literature or please contact npi electronic Software The normal experimental situation is the use of a com puter based data acquisition system for controlling the experiment Nevertheless all TEC systems are designed in a way that they can be used without software A stim ulus generator digital oscilloscope and chart recorder would be sufficient for working with these amplifiers see Fig 4 All TEC system s can be used in conjuncti on with the various software packages commercially available Input and output signals have calibrations that are suitable for m ost data acquisition packages They also provide special features such as electronic rem ote selection of m odes of operation and m onitor telegraph signals for the position of current gain and filter switches Th
34. e bandwidth of the current source which perform s the charge injection into the cell can be lim ited to 10 Hz by use of the BANDW Switch located on the current headstage In this m ode the cl amp circuit is capable of following only slow changes i e to keep the steady state IMPORTANT The controller m ust be used in P m ode INTEGRATOR OFF since parasitic oscillations m ay occur due to the limited bandwidth of the current source two integral components in a closed loop form an oscillator see ref 5 for details 13 WARNING If the bandwidth of the current headstage is set to 10 Hz som e functions such as RCel current electrode resistance test do not work properly Improvement of the Control Properties Control circuits with negative f eedback tend to be instable as a result of delays immanent in the system e g low pass characteristics of the microelectrodes or positive feedback caused by capacitive couplings between the electrodes With voltage clam p system s the control properties can be substantially im proved by shie Iding the electrodes from each other Often the shielding of the potential electrode suf fices to reduce the coupling capacity between the electrodes This shield can be connected to th e output of the buffer am plifier driven shield arrangement see Fig 1 The correct setting of the C com pensation increases the speed of response of the control loop but also increases the noise The correct setting of t
35. e handled with care Turn power off if headstages are connected or disconnected from the connectors on the front panel of the 19 cabinet TEC systems shall be used only in a warmed up condition to avoid temperature related errors Before using the TEC system s the output bias current of the current injection headstage m ust be canceled The tuning procedure is describe d in chapter 4 INSTALLATION Adjustm ent of the Current Offset CURR OUTPUT OFFSET Prevention of Line Interference It is recom mended that all experim ent should be carried out in a shielded environm ent Faraday cage connected to ground Such syst ems are described in the literature e g see refs 1 10 and 17 All com ponents inside this shielded area such as m icroscopes micromanipulators etc must also be grounded pr operly All electric systems in this area such as lamps perfusion valves electrical micromanipulators etc Are sources of noise which m ay deteriorate the measurement All TURBO TEC instrum ents have a high quality toroid transf ormer to keep down stray fields In spite of this noise problem s could occur if other m ains operated instrum ents are used The internal system ground GND socket should be connected to only one point on the measuring ground and should originate from the potential headstage The casings of the headstages are grounded and could be used to make this connection Multiple groundings which m ay form so called ground loops
36. ed Therefore the digital display is switched to the potential output of the current electrode SELECT switch to the left of the upper digital disp lay and the display is set to zero with the potentiometer Ce OFFSET Next a resistance of 1 10 MQ is connected from the current headstage output to ground as if an electrode were attached or the cell model is switched in the ON position The digital display and the CURRENT ELECTRODE potential connector Ce POTENTIAL OUTPUT x10m V now show a voltage deflec tion which is proportional to the flowing output current The output bias current can be tuned to zero with the C HEADSTAGE BIAS CURRENT control The current is zero when the voltage deflection is zero As a rule the current outputs CURR OUTPUT UNCOMPENSATED CURR OUTPUT and the CURRENT DISPLAY lower digital display should also read 0 uA This calibration can also be m ade during an e xperiment since no electrode is necessary and the potential reading is not affected Test of the Current Clamp Mode First the appropriate cell m odel is set up followed by the zero adjustm ent described above After this the offset of the potential electrode is set at zero If this adjustment is not possible it is an indication that the input am plifier in the headstage has been dam aged by electrostatic charge After the offset adjustment the function of the current clamp can be tested By application of a holding potential of for exam
37. eir input and output features as well as the m onitor telegraph signals provided allow very com fortable inter action between the clam p instrum ent and the data acguisition package If the software CellWorks available from npi electronic is used some of the functions of the TEC systems can be controlled directly from the computer 2 SYSTEM DESCRIPTION 2 0 GENERAL DESCRIPTION The TURBO TEC instruments are voltage current clamp systems which function according to the classic dual m icroelectrode m ethod This m ethod uses one m icroelectrode for the registration of m embrane potential and one for cu rrent injection The eguivalent circuit of a TEC system and the associated block diagram in VC mode are shown in Fig 1 and Fig 2 A view of the TEC front panel is given in Fig 3 Each amplifier of the Turbo TEC series ism ade up of a 19 basic system with a built in power supply and two m easuring headstages A smaller one for potential recording and one for current injection and recording All TEC system s are based on m odern state of t he art electronic circuits Their advanced design makes them superior to other am plifiers Some of the special features of TEC system s are differential potential registration high voltage current source output both to elim inate artifacts induced by the use ofm icroelectrodes TEC system s have autom ated electrode resistance test modes which can be used even with the electrodes impaled in a cell see
38. em ent with a time constant Tm which is always in the range of hundreds of milliseconds 23 In comparison with this physiological tim e constant the electronic tim e constants of the feedback loop can be considered as sm all and added to an equivalent tim e constant Te The ratio of the small and the large time constant determines the maximum gain which can be achieved without oscillations and thus the accuracy of the clam p With the gain adjusted to this level the integrator tim e constant and small tim e constant determ ine the speed of response of the system Tuning of the clam pis perform ed according to optim ization rules The absolute value optimum A VO provides the fastest response to a com mand step with very little overshoot maximum 4 while the sym metrical op timum SO has the best perform ance compensating intrinsic disturbance signals The SO shows a considerable overshoot maximum 43 to a step com mand which can be reduced by adequate shaping of the command pulse by a delay unit 5 and 13 An empirical tuning procedure is given in APPENDIX 2 The upper speed lim it is determ ined by the m aximum am ount of current which the clam p system can force through a given electrode see APPENDIX 3 The clam p performance can be increased considerably if th e influence of the current injecting electrode is excluded as far as possible from the clamp loop since the electrode resistance is nonlinear This is achieved
39. ent source has a high impedance floating output Therefore the zero position i e the zero of the bias current of this device has to be defined Since the used high voltage FET am plifiers become worm from the internal heat dissipation and their characteristics are strongly tem perature dependent the calibration procedure has to be done periodically by the user 20 The tuning procedure is done using the C HEADSTAGE BIAS CURRENT control and a resistance of a few MQ It is based on Ohm s Law the voltage deflection caused by the output current generated by the headstage on a test resi stor is displayed on the digital m eter The output current which is proportional to the m onitored voltage deflection is nulled with the C HEADSTAGE BIAS CURRENT control This tuning procedure cannot be performed with an electrode since there always are unknown offset voltages involved tip potential junction pot entials etc Therefore a test resistor of 1 10 MQ must be used If the TEC Cell m odel is used see Fig 6 only the C e and GND ground connectors m ust be connected The ON OFF GND switch can be used for the nulling procedure described below First the connection to the current electrode must be grounded This can be done directly with the wire connected to the C er connector or if the TEC Cell Model is used by switching the cell model in the OFF GND position Now the offset potential of the CURRENT ELECTRODE POTENTIAL output can be null
40. equency m onitor 8 7 V 1 V switch position output impedance 250 TEC 10CX f our pole lowpass Bessel f ilter with 16 corner frequencies 20 Hz 20 kHz frequency monitor 8 7 V 1 V switch position output impedance 250 CURRENT CLAMP TEC 10 standard current headstage Inputs 1 pA V 0 1 A V with ON O FF switches input resistance gt 100 k Q HOLD X XX nA ten turn digital control with 0 switch maximum 10 A For TEC 05X see separate sheets Noise potential output 100 uV pp current output 200 pA pp with 1 M resistance and 10 kHz bandwidth internal four pole Bessel filters Speed of response 1 settling tim e potential out put signals after application of square pulses of 1V with 1 M Q electrode resistance potential electrode lt 10 us current electrode lt 50 us VOLTAGE CLAMP Inputs two inputs with ON OFF switches sens itivity 10 m V and 40 m V input resistance gt 100 kQ HOLD XXX mV ten turn digital control with 0 switch maximum 1000 mV RISE TIME LIMIT 0 0 2 ms GAIN 10 A V 10000 HAN ten turn linear control INTEGRATOR TIME CONSTANT 200 us 2 ms ten turn control OUTPUT CURRENT LIMIT 0 100 linear control NOISE filters set to 10 kHz other settings see below Potential output lt 100 uV pp current output lt 10 nA pp at 10 kHz lt 2 nA at 500 Hz SPEED of RESPONSE VC Mode 1 settling time lt 80 us for 10 mV step and lt 100 us for 100 mV step applie
41. h e Marine Biological Laboratory Helsing r Denmark Polder H R and K Houam ed 1994 A New Ultra High Voltage Oocyte Voltage Current Clamp Amplifier in Elsner Nand H Breer eds G ttinge n Neurobiology Report 1994 Thieme Verlag Stuttgart Polder H R R Schliephacke W St hm er and H Terlau 1997 A new switched m ode double electrode clam p am plifier avoiding series resistance errors in Elsner N and H Wassle eds G ttingen Neurobiology Report 1997 Thieme Verlag Stuttgart Rudy B amp L E Iverson eds 1992 Ion Ch annels Section II A Expression of lon Channels in Xenopus Oocytes Methods in Enzym ology Volume 207 Academ ic Press San Diego Schoepfer R A F ll and H R Polder 1996 EggWorks A New Control Software for the Entire Experimental Setup in Elsner N and H U Schnitzler G ttingen Neurobiology Report 1996 Thieme Verlag Stuttgart Smart T G and B J Krishek 1995 Xenopus Oocyte Microinjection and lon Channel Expression from Boulton A A Baker G B and W alz W eds Patch Clam p Applications and Protocols Neuromethods Vol 26 Humana Press Totowa New Jersey Smith T G Jr Lecar H Redm ann S J and Gage P W eds 1985 Voltage and Patch Clamping with Microelectrodes American Physiological Society Bethesda The W illiams amp Wilkins Company Baltimore St hmer W 1992 Electrophysiological Recording from Xenopus Oocytes in Rudy B amp L E I
42. he Transient Compensation TEC 10CX This test should be perform ed after com pletion of the voltage clam p mode test W ith the application of test pulses itm ust be possibl e to suppress the capacitive current to a large extent with the regulators A1 A3 and T1 T3 The linear leakage current which flows through the resistance of the cell m odel must be compensated by the LINEAR potentiometer Note that the BYPASS ON switch has tobe in ON position in order to use the transient compensation Oscillation Shut Off First set the DISABLED RESET switch in the DISABLED position green light will light Then set the switch in the m iddle position a nd overdrive the C com pensation to cause oscillations to appear The response threshold can now be adjusted with the THRESHOLD potentiom eter If the system responds the LED will light red and the current injection and C com pensation are shut off In order to adjust the C com pensation to normal the system can be restored by switching to the RESET position After successful com pletion of these adjustm ents the instrum ent is ready for use For experimental m easurement follow the sam e order of procedure adjustm ent of the offset compensation in the bath pre adjustm ent of the C com pensation and of the shut off and further adjustments after the positioning of the electrodes 22 5 CONTROL THEORY APPENDIX 5 1 THEORY OF OPERATION OF THE TEC SERIES AMPLIFIERS The standa
43. he different parameters results in a com promise between the stability accuracy noise and control speed Adjustment criteria speed of response and lin earity are discussed in the Control Theory Appendix 5 2 6 ADDITIONAL SYSTEMS Audio Monitor The membrane potential or the potential of the cu rrent electrode can be translated into an acoustic signal voltage to frequency conversion Not all instrum ents are equipped with this device The signal from both electrodes can be connected to the AUDIO MONITOR the selection is performed by a toggle switch The volume can be set by a potentiom eter located on the left side of the front panel Oscillation Shut Off This system shuts off the current injection a nd the C com pensation if oscillations that m ight damage the preparation appear This m ay happen if the capacity com pensation is set at too high levels or if the voltage clamp gain is too high The threshold at which the shut off system is activated can be set in advance with a potentiometer THRESHOLD The correct setting must be found by trial and error A green light shows the correct function of the amplifier i e normal operation is possible a red one shows when it has shut down If the red light is on the system must be reset with the DISAB LE RESET switch In the DISABLED position the shut off function is turned off green LED on WARNING If the red light is on only the electrode resistance test and the potential regi
44. imum current in voltage clamp mode VC GAIN Proportional amplification of the PI controller VC ERROR Display of control error SERIES RESISTANCE COMPENSATION ON switc h Adjustm ent of series resistance compensation cur prop amplification AUDIO MONITOR Monitors the potential signals POWER Power switch TEC 05X option Optionally the TEC 05X is equipped with a BUZZ function to facilitate penetration of the cell membrane BUZZ operation is based on overcom pensation of the respective electrode The electrode for BUZZING is selected via the ELECTRODE RESISTANCE switch BUZZ Push button to activate BUZZ DURATION Potentiometer to set the duration of the BUZZ TTL REMOTE BNC connector for connecting a remote switch TTL high BUZZ 3 2 REAR PANEL FUSE 0 63 A 220V 1 25 A 110V SLOW Mains fuse 115 220V AC Mains cable hook up through an IEC standard plug INTERNAL GROUND System ground PROTECTIVE EARTH Mains ground CURRENT OUTPUT SENSITIVITY MONITOR 1V STEP 1 to 7V signal m onitoring the position of the CURRENT OUTPUT SENSITIVITY switch CURRENT FILTER MONITOR 1V STEP 7 to 8V signal monitoring the position of the CURRENT FILTER switch 17 3 3 POTENTIAL HEADSTAGE Pg Electrode connector with driven shield REF Connection of the reference electrode for measurement of the bath potential GND Ground connector TEC 05X option The TEC 05X is equipped with a bridge m ode With the electrode connected to
45. ion of basic set up co_nstruction and necessary equipment please consult the literature Chapter 6 refs 1 10 11 and 21 23 The stimulator or DAC output of the data acquis ition system is connected either to the one of the CURRENT STIMULUS INPUT BNCs or the VOLTAGE COMMAND BNCs Two input channels of the scope or ADC inputs of the data acquisition system are connected to the CURRENT OUTPUT respectively POTENTIAL OUTPUT of the TEC For remote operation through TTL signals or from the computer system connect TTL signals or control cable to the MODE of OP ERATION selection BNCs see 2 7 DIGITAL CONTROL UNIT Connect headstages to electrodes or cell model as outlined in figs 5 and 6 TEC Cell Model The testing of the TURBO TEC systems should be perform ed with appropriate cell m odels see Fig 5 and Fig 6 For all tests ex cept the CURRENT HEADSTAGE BIAS TUNING procedure the TEC Cell Model must be set ON We recommend the 100k m embrane resistance see Fi g 6 and the use of square test pulses of 1 V This corresponds to a com mand of1 uA in CC m ode and to 100 m V steps in VC mode Adjustment of the CURRENT HEADSTAGE BIAS CURRENT control This tuning procedure is very important since it determines the accuracy of the TEC system TEC system s are equipped with a high voltage current source which is connected to the current injecting electrode and perform s the current injection see SYSTEM DESCRIPTION chapter 2 2 This curr
46. is split into two controls Coarse controls at the headstag es and a fin e controls at the front p anel of the am plifier The coars e controls are used to set a range for the fine controls Esp ecially in VC m ode the coars e controls are very sensitive and can cause oscillations if overcompensated Note The electrode res istance is dis played correc tly only if the ca pacity for the res pective electrode is compensated properly Removed Front Panel Element BATH POTENTIAL BNC connector is not installed MODE Selection Mode of Operation Current Injection Current Remarks Signal DHC Dynamic Hybrid Clamp not implemented EXTERN via MODE SELECT From CURRENT HEADSTAGE CC or VC INPUT BNC with TTL VC VOLTAGE CLAMP From CURRENT HEADSTAGE OFF No current injection Electrode resistance test works CC CURRENT CLAMP From CURRENT HEADSTAGE two electrode MODE BRIDGE CURRENT CLAMP From POTENTIAL HEADSTAGE in BRIDGE mode Note The EXTERN mode is exclus ively for the two electrode m odes CC and VC It is not possible to activate the BRIDGE mode remotely Scaling as labeled at the front panel CURRENT OUTPUT FROM HEADSTAGE 0 1V nA CURRENT STIMULUS INPUT 1nA V CURRENT OUTPUT SENSITIVITY 0 1 V nA to 10V nA HOLDING CURRENT X XX nA 1 e 150 is 1 50 nA ELECTRODE RESISTANCE determined using 1 nA test pulses approx 150 Hz XXX MQ Capacity Compensation First part basic setting
47. l Model are available on request Please refer to chapter 7 for details or contact npi electronic for details 2 1 POTENTIAL REGISTRATION Arrangement of the Recording Electrodes For m embrane potential registration all TEC amplifiers use a differential electrode arrangement to record the membrane potential as accurately as possible Fig 1 and Fig 5 A description of the potential headstage can be found in chapter 3 3 1 Two electrodes an intracellular m icroelectrode P gr potential electrode and an extracellular electrode REF reference electr ode which are connected to high im pedance buffers input resistance better than 10 Q in the potential headstage are required for potential measurement In addition the bath surrounding the cell m ust have a severe ground connection Ag AgCl pellet or Agar bridge s ee Fig 5 which can carry the large m embrane currents flowing during voltage clam p experim ents This arrangem ent ensures the m ost accurate m easurement of the transm embrane potential the reference electrode REF measures the bath potential extracellular potential which is subtracted from the intracellular potential recorded by the intracellular electrode P gr Intracellular m icroelectrodes used for oocytes have resistances of 300 k O upto 1 2 M Q the resistance of the REF electrode is usually much smaller a few ten KQ maximum Capacity Compensation The frequency response of the potential electrode low
48. lectrode which is monitored at the display is selected by a toggle switch located at the left side of the display POTENTIAL ELECTRODE or CURRENT ELECTRODE In addition the recorded potentials are converted to a sound with a potential dependent pitch with the AUDIO MONITOR The electrode which is monitored by the AUDIO MONITOR is also selected by toggle switch POTENTIAL ELECTRODE or CURRENT ELECTRODE 2 2 CURRENT INJECTION AND CURRENT MEASUREMENT Current Injection The current injection is perform ed by means of a glass microelectrode which is connected to the current headstage CeL A description of the current headstage is given in chapter 3 2 The unique advantage of the instrum ents in the Turbo TEC series is the voltage controlled current source output V C or V I converter fo r electrical com pensation of the disturbances from the microelectrode during current injecti on i e high resistance and stray capacity see Polder 1984 Polder amp Swandulla 1990 This current source is built into the current headstage Due to this current source output current injection becom es independent from the resistance of the m icroelectrode which is usua lly strongly nonlinear In addition this circuit allows direct measurement of the current injected in the cell without the necessity of a virtual ground circuit for the bath Output Current Zero C HEADSTAGE BIAS CURRENT In order to adjust the zero current of the out put current source each
49. lectrodes TEMPERATURE DRIFT WARM UP TIME All analog electronic systems are sensitive to temperature changes Therefore all electroni c instruments containing analog circuits shall be used for recordings only in a warm ed up condition i e after internal tem perature has reached steady state values In most cases a warm up period of 30 minutes is sufficient 1 INTRODUCTION About this Manual This instructions manual describes the most important functions and operation possibilities of the TURBO TEC 05 and TURBO TEC 10 fam ily of Voltage Current Clam p amplifiers A short introduction to the theory and practice of the voltage clamp and current clamp technigue is also included as far as it is necessary fo r understanding the operation of this instrum ent A broad selection of literature of which we give a selection at the end of the m anual is available on these techniques The manual is divided into 8 chapters 0 7 Chapter 0 Safety Regulations gives som e hints for the safe operation of the instrum ent Following this chapter 1 Introduction in the chapter 2 System Description the functioning of the device is outlined followed by chapter 3 Controls and Connectors which describe s the control switches and displays The Installation chapter 4 prescribes the calibration and test procedures prior to and at the onset of an experim ent The Control Theory Appendi x chapter 5 describes som e theoretical aspects and provi
50. pass characteristic is compensated for by a feedback circuit negative capacity compensation CAPACITY COMPENSATION 10 turn potentiometer and a driven shield a rrangement for an overview see Ogden 1994 The dial is not calibrated and has its zero position on 000 Since in oocyte experiments microelectrodes are usually in the one MQ range or below for most experiments it is not required to use capacity compensation WARNING Capacity com pensation is based on positive f eedback Theref ore overcompensation causes oscillations ringing wh ich can deteriorate the preparation or the recording electrodes Therefore the control m ust be handled with care and before im paling a new cell must be set to 000 Offset Compensation All microelectrodes produce a potential by them selves the tip potential This nonlinear and must be com pensated electronically The tip potential of the potential registration microelectrode P g is equalized for the m ost part autom atically through the differential potential registration The remaining offset is compensated manually by adjusting the offset com pensation controls which is available for each electrode 10 turn potentiom eters POTENTIAL OFFSET approximately 200 mV and CURRENT ELECTRODE OFFSET approximately 500 mV WARNING Of fset controls are sym metrical operating both in positive and negative direction therefore setting to zero occurs on position 5 00 on the dial 10 ism aximum in po
51. ple 1 UA the membrane resistance m ust result in an appropriate change in potential according to Ohm s law Test of the Voltage Clamp Mode After the test of the current clam p mode the function can be switched to the voltage clam p mode The control param eters are best adjusted by application of a te st pulse see Appendix 2 Empirical Tuning Procedure After this the current is measured relative to a given holding potential for example 100 mV The measured current should correspond with one calculated by Ohm s law 21 Testing of the Zero Current Zero Potential After completing these three tests all input signa ls should be shut off All potentials and the current should equal zero in both the current clamp and in the voltage clamp mode Resistance Measurement Test the resistances of the m icroelectrodes by first switching the MODE OF OPERATION key to the appropriate test system TEC 10CX or by switching the ELECTRODE RESISTANCE switch to the desired position The function of this system is tested with the most accurate resistances possible These systems function independently of the other adju stments with the condition that a connection between the electrodes and ground connector bath exists If an appropriate display does not appear relative to a given resistance it is an indication that the input am plifier of the respective m easuring headstage has been dam aged by electrostatic charge Adjustment and Test of t
52. ra G Rom ano A Tiso N Pe ric M Maffia M Boll M Argenton F Daniel H amp Storelli C 2003 Molecular and functional characterisation of the zebrafish Danio rerio PEPT1 type peptide transporter FEBS Lett 549 115 122 30 7 TURBO TEC SERIES SPECIFICATIONS All following current signal related param eters are for the TEC 05 and TEC 10 instrum ents with standard 150 V current headstage Parameters for the other system s or for syst ems with a selectable current ranges can be calculated from these parameters MODES of OPERATION DHC Dynam ic Hybrid Clam p Mode TEC 05 option CC Current Clam p Mode VC Voltage Clamp Mode OFF Mode BRIDGE Bridge Mode option TEC 05 MODE selection 6 position toggle switch TEC 05 or pushbuttons TEC 10 LED indicators remote selection by TTL inputs ELECTRODE RESISTANCE test POTENTIAL ELECTRODE m easurement of the ELECTRODE RESISTANCE of the POTENTIAL ELECTRODE CURRENT ELECTRODE m easurement of the ELECTRODE RESISTANCE of the CURRENT ELECTRODE HEADSTAGES TEC 10 Potential headstage Differential input for suppression of bath potentials cmr gt 80 dB Input resistance gt 10 operating voltage 15 V Electrode connector BNC with driven shield driven shield range 15 V output im pedance 250 Q Reference connector bath gold plated SUBCLIC grounded shield ground connector 2 3 mm connector or headstage enclosure Size 65x25x25 mm headstage enclos
53. ransient Compensation TEC LOCK WEE 10 e 10 OPUONS masky casu tka A ON a aa al io lud ua 11 2 3 ELECTRODE RESISTANCE MEASUREMENT ono noonnnonnnnnnnnnn 11 2A CURRENT CLAMP MODE CO a al la 12 AER E 12 2 5 VOLTAGE CLAMP MODE VC obdo da 12 EE 12 e EE 12 Control Circuit D Teontroaller nono no nnononnnnn non ocn ono rcononnnno nn anno 12 VEO Display ai LA A r iol 13 Current Limit VC OUTPUT LIMIT E 13 Series Resistance EE 13 POW e Mode tdi na outs 13 Improvement of the Control Properties s sssistisisstrasnebtula skody sla a 14 20 ADDITIONAL SYSTEM Suust ee Eeer 14 Audio Monitor ie la a li 14 EE GE Eeer 14 2 DIGITAL EN CHE UNI tt A 15 Mode of operation SE CC A E 15 3 CONTROLS AND CONNECTORS seen 16 3 1 FRONT PANEL A A a ao 16 TECSI OPUS tds ooo ld 17 3 2 REAR PANEL nnee iti aai za ui 17 3 3 POTENTIAL HEADS TAGE EEN 18 RE SE EE 18 sA GURRENTHEADST AGE ss A eS ee o 18 SANSTALLATION BET 19 4 1 GENERAL CONSIDERATIONS i sa a 19 RE O O 19 Prevention of Line Interference geegent eebe 19 4 2 TESTS AND TUNING PROCEDURES tada 20 General Considerations ia aia da A o A lab ad 20 BASIC connections LEA NL ee EE 20 TEC E is ho ta dat lata vata aa a saat 20 Adjustment of the CURRENT HEADSTAGE BIAS CURRENT contra 20 Test of the Current EE 21 Test of the Voltage Clamp Mode eege 21 Testing of the Zero Current Zero Potential A 22 Resistance VICE EE hala otras ab r A 22 Adjustment and Test of the Transient Compensation TEC TOCK
54. rd configuration for voltage clam ping oocytes is the two electrode voltage clam p arrangement 19 23 In contrast to previously described clamp systems for review see ref 11 and 20 the instruments for oocyte clamping must meet special requirements since oocytes are very large cells with a high m embrane capacity up to 100 500 nF and large m embrane currents up to 100 LA and more Voltage clamp instruments are closed loop cont rol systems with two inputs which act from outside on the control loop An electronic feedback network is used to force the m embrane potential of a cell to follow a voltage com mand setpoint input as fast and as accurately as possible in the presence of incom ing disturban ces disturbance input correlated with the activities of the cell by injecting an adequate amount of charge The current injected by the clamp instrument is a direct measure for the ionic fluxes across the membrane see references 4 9 11 and 20 The perform ance evaluation and optim al tuning of the system can be done by considering only the com mand input since the m athematical models set point transfer function and the disturbance transfer function see 5 and 10 13 are closely related Modern control theory provides adequate solutions for the design and optimal tuning of feedback systems 5 Most voltage clamp systems are composed only of delay elements i e elem ents which react with a retardation to a change This type of closed loop s
55. rode two outputs sensitiv ity x10 mV and x40 m V output im pedance 250 Q output voltage range 15 V Current electrode sensitivity x10 mV output impedance 250 output voltage range 15 V DISPLAY switch selected XXX mV AUDIO MONITOR Pitch correlated with potential signals switch selected OSCILLATION SHUT OFF Turns off current injection and capacity com pensation function displayed by red green LED disabled off reset switch threshold set with linear control 0 1200 mV ELECTRODE RESISTANCE TEST both electrodes 100 mV MQ obtained by application of square current pulses 10 nA display XX X M Q selected automatically CURRENT OUTPUTS Uncompensated output signal sensitiv ity 0 1 V UA output resistance 250 Q output voltage range 15 V Compensated filtered output sensitivity 0 1 10 V uA in 1 2 5 steps selected by rotary switch with lowpass Bessel filter output im pedance 250 Q sensitivity monitor 1 7 V 1V switch position output impedance 250 Q DISPLAY X XX pA 32 CURRENT SIGNAL PROCESSING TEC 10 transient compensation unit with three overlapping ranges max T1 3 3 ms T2 330 us T3 33 us tim e constants set with te n turn controls am plitudes set with one turn linear controls leakage compensation maximum 1 LA CURRENT OUTPUT FILTERS TEC 05X two pole standard version orf our pole lowpass Bessel f ilter TEC 05X BF system with 16 corner frequencies 20 Hz 20 kHz fr
56. s G ttingen Neurobiology Report 1997 Thieme Verlag Stuttgart Greeff N G and H R Polder 1998 Optim ization of a two electrode voltage clam p for recording of sodium ionic and gating curre nt from Xenopus oocytes Biophysical Society Meeting ThPos 238 Biophysical Journal supplement Greeff N G F J P Kuhn and W Kathe 1998 Gating Currents reveal hidden rat brain IIA sodium channel expression in Xenopus oocytes Biophysical Society Meeting TU PM P2 Biophysical Journal supplement Jack J J B Noble D and Tsien R W 1975 Electric Current Flow in Excitable Cells Claredon Press Oxford Kettenmann H amp Grantyn R eds 1992 Practical Electrophysiological Methods W iley Liss New York Ogden D ed 1996 Microelectrode Techniques The Plym outh W orkshop Handbook Second edition The Company of Biologists Ltd Cambridge Polder H R 1984 Entwurf und Aufbau eines Ger tes zur Untersuchung der Membranleitfahigkeit und deren Nichlinearit t nach der potentiostatischen Methode Voltage Clamp Methode m ittels einer Mikroelektrode Diplom arbeit M Sc Thesis Technical University Munich Polder H R and Swandulla D 1990 Desi gn and Optim al Tuning of Single and Double Electrode Voltage Clam p System s Using Met hods of Modulus Hugging Pfl gers Archiv 415 S77 28 Polder H R 1993 Voltage and Current CI amp Methods in Cellular Signalling Course Book of the European Sum mer School att
57. s using the respective potentiometers at the front panel Immerse both electrodes into the bath Compensate for the OF FSETs of both elect rodes using the respective potentiometers at the front panel Carefully compensate the capacity of both electrodes using coarse and fine controls Apply square test pulses at CURRENT STIMULUS INPUT connector Switch the operation MODE selector to BRIDGE Watch the POTENTIAL OUTPUT P gy at the oscilloscope and ad just the BRIDGE BALANCE using the BRIDGE BALANCE po tentiometer After adjustm ent you should see a straight vo ltage trace without artifacts caused by the potential drop at the electrode resistance Disable all CURRENT STIMULI CUR RENT STIMULUS INPUT an d HOLDING CURRENT Find a cell and approach the cell membrane with the POTENTIAL ELECTRODE Penetrate the cell m embrane with the POTENTIAL ELECTRODE If you are not successful try the BUZZ function If the electrode tip is insi de the cell you should read the mem brane potential at the DISPLAY the POTENTIAL DISPLAY switch benea th the DISPLAY set to POTENTIAL ELECTRODE You are now ready to apply test pulses to the cell BRIDGE mode Two electrode operation Set the POTENTIAL DISPLAY swi tch to CURRENT ELECTRODE a nd switch the operation MODE selector to CC Approach the cell membrane with the current electrode Penetrate the cell membrane If you are not successful try the BUZZ function If the electrode tip is insi de the
58. s necessary for the ad equate shaping of the current input signal COMMAND INPUT All instrum ents are equipped for the injection of a constant current HOLD control X XX uA adjustable through a 10 turn potentiometer with a digital display and with an analogue input The polarity is controlled by a switch with which the HOLD current signals can also be turned off Current Clamp Inputs The inputs are analogous to those of the voltage clamp mode A constant holding current is set on the 10 turn HOLDING potentiom eter with a LA display X XX pA i e m ax range is 9 99 uA The polarity of the HOLDING control is controlled with the 0 switch In the 0 position the HOLDING control is turned off Th e analogue current input is calibrated with 1 UA V i e 1 V generates a current of 1 pA This input is controlled by an ON OFF switch 2 5 VOLTAGE CLAMP MODE VC In the voltage clam p mode the m embrane potential is forced by a controller to m aintain a certain value or to follow an external co mmand which allows m easurement of ion fluxes across the cell m embrane independent of potential changes and separate f rom capacitive current flows This is the m ost complex mode of operation with these instrum ents Special precautions must be taken while tuning the cont rol circuit in order avoid stability problem s IMPORTANT Although in VC m ode one is prim arily interested to record the current flowing across the m embrane the clam p circuit con
59. sitive direction 0 is maximum value on negative direction Current Electrode Potential Recording In order to determ ine whether both electrodes are inserted into the sam e cell the potential of the current injecting m icroelectrode C gr see 2 2 is recorded by a buffer am plifier in the current headstage with a x10m V scaling C el POTENTIAL x10 m V BNC This unit is equipped with an offset compensation CeL OFFSET ten turn potentiometer 500 mV WARNING Due to the lim ited operation range of the output am plifier 12 V m aximum the high voltage signals occurring during curre nt injection will drive this output into saturation clipping Therefore during electr ode positioning no current flow should occur through the current electrode Cg During voltage clamp mode the signals at this output may become very noisy WARNING The Ce Offset control is sym metrical operating both in positive and negative direction therefore setting to zero occurs on position 5 00 on the dial 10 ism aximum in positive direction 0 is maximum value on negative direction Potential Monitor and Audio Monitor The measured membrane potential is amplified Pe by a factor of 10 or 40 Cer by a factor of ten The recorded potentials from both microelectrodes De and Cr can be read out from the respective BNC sockets POTENTIAL OUTPUT P gr x10 or P py x40 and C gy x10 and can also be directly not am plified displayed in mV on a digital display The e
60. stration and display unit work All other func tions of the am plifier are shut of f i e it cannot be used any more for VC or CC recordings In the DISABLED position the green light is always on i e all amplifier function are activated if oscillations occur the preparation may be damaged 14 2 7 DIGITAL CONTROL UNIT All signal in the TEC instrum ents are select ed and com muted by electronic devices analog switches and multiplexers which are controlled with digital signals This allows synchronous switching procedures that avoid switching artifacts and facilitates the use of the instrument Many functions can also be controlled by com puter signals A digital control interface is available Please contact npi electronic for details Mode of operation selection TEC 10CX systems The selection can be made manually or remotely using TTL signals The selected function is displayed by LED s On TEC 10CX systems the selection of the four operation modes Rce CC VC Bea can be made with push buttons with LED display Remote selection can be m ade with TTL trigger signals applied to the respective inputs rear panel Software selection optional TEC 10CX CW from the CellW orks software package see Chapter 1 W ith the ON EXTERN switch located below the current f ilter the selection between front panel control ON and software control EXTERN can be selected On TEC 05X systems a 6 position switch is used for the selection of
61. tential recording and feedback electronic sy stems Therefore the design of the potential recording site is very important A differential potential registration with a reference electrode which registers the bath potential m inimizes errors due to resistances in series with the cell membrane Driven shield and capacity compensation circuits are used to improve the speed of response In some cases a series resistance compensation circuit which adds a current proportional gain can improve the clamp performance considerably 6 The use of such a circuit enhances the speed of response and im proves the accuracy of the clam p system Since both circuits are positive feedback loops the noise level is also increased 24 In addition to the elem ents of the clam p loop itself the oocyte clam p amplifier needs som e additional units which f acilitate the work such as electrode resistance test units oscillation shut off unit adequate output signal am plification filtering and display units facility for compensating the capacitive currents etc EMPIRICAL TUNING PROCEDURE FOR PI CONTROLLERS Before switching to voltage clam p mode all pa rameters related to the recording electrodes offset capacity com pensation etc have to be tuned in CC m ode With PI controller based clamps capacity com pensation tuning can be rep eated whenever it is necessary also in VC mode Before switching to VC mode gain control has to be reduced to a safe level integra
62. tive currents in the voltage clam p m ode CURRENT TRANSIENT COMPEN SATION For the TURBO TEC 05X the com pensation unit is available as a separate instrument for the modular EPMS 07 system TRC 01M Through this compensation unit the voltage clamp pulse is differentiated with three adjustable time constants and added to a linear com ponent to com pensate for the leakage current This signal is subtracted from the m easured curre nt signal The adjustm ent is m ade through 4 potentiometers for the amplitudes A 1 A2 A3 and linear as well as 3 10 turn potentiom eters T1 T3 for the tim e constants of the differe ntiators A BYPASS switch allows to quickly switch off the compensation except the linear component Current Filter A tunable low pass CURR FILTER is attached to this amplifier The TEC 10CX has a four pole low pass Bessel f ilter with 12 or 16 corner frequencies 20 50 100 200 300 500 700 1k 1 3k 2k 3k 5k 8k 10k 13k 20k Hz The position of the switch is monitored by the FREQUENCY M ONITOR signal 5 6V 1V step 12 position filter and 8 7V 1 V step 16 position filter In the TEC 05 itm ay be a single pole oraf our pole Bessel f ilter with 12 or 16 corner frequencies see TEC 10CX 10 Options Some of the Turbo TEC instrum ents have curre nt headstages with four ranges see also page 10 The TEC 10 m odel can also be purch ased without transient com pensation Please ask npi for details 2
63. tor has to be switched off to increase stability The PI controller is first used as P controller only INTEGRATOR switch in OFF position The command input is used without sm oothing Identical com mand pulses are applied The gain is increased until the overshoot of the de sired tuning method appears Using only the P part of the controller means that a steady state error will be present Now the I section is reconnected to form the PI controller INTEGRATOR in ON position The integrator tim e constant is set to give the desired overshoot according the optim ization rules of Appendix 1 If the SO is used an external com mand input filter has to be used to reduce the overshoot according to the requirements of the experiment 5 3 SPEED OF RESPONSE AND LINEARITY OF THE CAPACITIVE TRANSIENTS For the investigation of voltage activated ch annels with voltage clam p instrum ents som e special techniques for eliminating the capacitive and leak currents have been introduced such as the P 4 ore more general P N protocol see 17 for overview For these protocols the speed and linearity of response of the clamp system is of great importance As outlined in APPENDIX 1 the TEC system sare designed f ollowing a control theory procedure called m odulus hugging see referen ces 5 12 16 The procedure requires a PI proportional integral controller This procedur e is applicable to control system s composed of an element with one large
64. trols primarily membrane potential The better the potential is controlled i e the sm aller the VC error signal com mand signal minus recorded signal can be m ade the m ore accurate on can record m embrane currents Som e theoretical aspects are presented in the Control Theory Appendix see also references Voltage Clamp Inputs The inputs are analogous to those of the current clamp mode A constant holding potential is set on the 10 turn HOLDING potentiom eter w itham V display The polarity of the HOLDING control is controlled with the 0 switch In the 0 position the HOLDING control is turned off There are two anal ogue inputs one is calibrated with 10 mV mV analogous to the x10 mV potential output a nd the second is calibrated 40 x0 025 corresponding to the x40 output The inputs ar e controlled with the respective ON OFF switches Rise Time Control With application of a pulse the m aximum ri se tim e can be lim ited with a control The calibration corresponds to a pulse of 100 mV 1 Von 10 mV command input The rise tim e limit is necessary to dam pen the overshoots while calibrating the control circuit according to the symmetrical optimum described in the Control Theory Appendix Control Circuit P I controller The TURBO TEC systems are equipped with Proportional Integral PI control loops These compare the measured membrane potential with the set command potential The difference is then amplified and integrate
65. ure is connected to ground Holding bar diameter 8 mm length 10 cm Current headstage high voltage Operating voltage range 150 V standard TEC 10 or 225 V TEC 225 system s input resistance gt 10 Q internally trim mable electrode connector gold plated SUBVIS connector grounded shield Power dissipation 6 W standard system or 20 W TEC 225 system Size 100x50x30 m m with heat sink 100x50x20 150V or 225x40x60 m mor equivalent TEC 225 grounded enclosure holding bar iso lated from ground standard system only diameter 8 mm length 10 cm Current range 150 uA 1 MQ TEC 10CX oocyte systems 220 uA 1 MQ TEC 225 Current range switch optional X2 x5 x10 or x0 1 x0 1 x0 2 x0 5 x1 Current headstage TEC 05 see additional information sheets 31 Bandwidth and Speed of Response Full power bandwidth Re 0 gt 100 kHz rise tim e 10 90 current pulse of 100 LA applied to Re 1 MQ lt 30 us Bandwidth switch wide band or 10 Hz for parallel patch clamp recordings Current Electrode Parameter Controls Leakage current adjustable to zero with ten turn control of fset com pensation ten turn control 1200 m V capacity com pensation optional TEC 05X range 0 30 pF ten turn potentiometer Potential Electrode Parameter Controls Capacity compensation range 0 30 pF ten turn control offset com pensation 200 mV ten turn control POTENTIAL OUTPUTS Potential elect
66. uring the experim ent especially after impaling a c ell it is ne cessary to fine tune the CAPACITY CO MPENSATION during the experiment using the CA PACITY COMPENSATION control at th e amplifier To get familiar with th is connect a cell m odel and go through the following steps the procedure is the identical with a real cell O Connect POTENTIAL OUTPUT P e and CURRENT OUTPUT front panel to oscilloscope U Set the HOLDING CURRENT to zero W ith the am plifier in CC m ode apply s mall square pulses to the cell Negative current pulses are recommended If you apply positiv e current pulses be sure only to elicit ohm ic responses of the cell m embrane i e pulses should not elicit openings of voltage gated channels OQ The POTENTIAL OUTPUT P r should show the ohm ic response of the cell m embrane without an artifact Important The electro de resistance test is accurate only if the capac ity if the e lectrodes is well compensated Bridge Balance If current is passed through an electrode the occurring voltage deflec tion potential drop at Re affects the reco rding of mem brane potenti al Th erefore th is de flection m ust be compensated carefully by means of the BRIDGE BALANCE control With the cell model connected or the electrode in the bath the BRIDGE BALANCE control is turned on clockwise until there is no artifact on the POTENTIAL OUTPUT Py J Connect a cell m odel or immerse the electrode into the bath as deep
67. verson eds 1992 Ion Channels Met hods in Enzymology Vol 207 Academic Press San Diego St hmer W Terlau H and Heinem ann S H 1992 Xenopus Oocytes for Two Electrode and Patch Clam p Recording in Kettenm ann H amp Grantyn R eds 1992 Practical Electrophysiological Methods Wiley Liss New York St hmer W and A B Parekh 1995 Recording from Xenopus Oocytes in Sakmann B and E Neher eds Single Channel Recording Second Edition Plenum Press New York and London Madeja M et al 1991 A concentration clam p system allowing two electrode voltage clamp investigations in oocytes of Xenopus laevis J Neuro Meth 38 267 269 Madeja M et al 1995 Im provement and te sting of a concentration clam p system for oocytes of Xenopus laevis J Neuro Meth 63 211 213 St hmer W 1998 Electrophysiologic Recordings from Xenopus Oocytes in P Michael Conn ed Ion Channels Part B Meth in Enzymology Vol 293 Academic Press San Diego Polder H R and D Swandulla 2001 The use of control theory for the design of voltage clamp system s A sim ple and standardized procedure for evaluating system param eters J Neurosci Meth 109 97 109 29 Kottra G and H Daniel 2001 Bidirectiona 1 electrogenic transport of peptides by the proton coupled carrier PEPT1 in Xenopus laevis oocytes its asym metry and sym metry J Physiol 536 2 495 503 Greeff N G and F J P K hn 2000 Variable Ratio of Perm
68. xperiment with the electrode in the tissue but not in a cell Electrode Selection Electrodes must be tested before use This is done by applying positive and negativ e current pulses and by com pensating with the BRIDGE BALANCE control Electrodes which show significant changes in resistance rectification cannot be used for intr acellular recordings By increasing the current am plitude the capability ofthe electrod eto carry current can be estimated The test current must cover the full range of currents used in the experiment Sometimes the performance of electrodes can be improved by breaking the tip or by using the BUZZ function of the amplifier overcompensated compensated undercompensated 5mY Vin 20 ms l m 1 NA 20 ms Figure 1 Adjustment of the bridge balance after penetrating a cell undercom pensated potential mV 0 50 100 150 200 250 300 350 400 time ms overcompensated potential mV A time ms compensated potential mV 3 2 si 1 4 D 1 2 4 3 1 i i i 1 0 50 100 150 200 250 300 350 400 time ms potential Figure 2 Tuning of the BRIDGE BALANCE using 100 MQ resistor A uncompensated potential mV current nA 100 30 80 25 60 20 40 15 20 10 D ER o5 40 00 160 B time ms Catray compensated potential mV current nA 80 Tor 30 70 Ki 25 60 50 20 40 Tem 15 30 20 Want 10 10 65 0 10 60
69. ystem s can be optim ized easily by adequate shaping of the frequency characteristic m agnitude F jw of the associated transfer function F s output to input ratio in the frequency dom ain LAPLACE transform of the differential equation of the system Using controllers with a proportional integral characteristic PI controllers it is possible to force the m agnitude of the frequency characteris tic to be as close as possible to one over a wide frequency range m odulus hugging see 5 and 12 15 This m eans that the controlled membrane potential rapidly reaches the desired command value The PI controller yields an instantaneously fa st response to changes proportional gain while the integral part increases the accuracy by raisi ng the gain below the corner frequency of the integrator i e for slow signals to very high va lues theoretically to inf inite for DC signals i e an error of 0 without affecting the noise level and stability Since the integrator induces a zero in the transfer function the clam p system will tend to overshoot if a step com mand is used Therefore the tuning of the controller is performed following optimization rules which yield a well defined system performance AVO and SO see below The various components of the clamp feedback electronics can be described as first or second order delay elements with time constants in the range of m icroseconds The cell capacity can be treated as an integrating el

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