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USE OF THE PATCH CLAMP – A TUTORIAL  

•

  37

Chapter 3

solution to which 120-150 µg/ml of Nystatin or Amphotericin B [from a stock
solution of 30 mg/ml in DMSO] has been added.  Over a 5-30 min. time period
these polyene antibiotics form myriad tiny cation-selective, voltage-independent
channels in the membrane patch.  These channels allow small ions to equilibrate
between the cell and the pipette allowing the cell to be voltage clamped through the
open channels.  Since substances as large as, or larger than, glucose will not
permeate these channels, cell contents are not washed out as in standard whole-cell
techniques.  This is an advantage or a disadvantage, depending on the experiment.
With this technique, a rise in whole-cell capacity transients will be observed as the
antibiotic partitions into the cell as shown in Figure 10.

400 pA

3 ms

1 MIN

2 MIN

3 MIN

4 MIN

5 MIN

Figure 10.

   Going whole-cell:  capacity transients observed during amphortericin partitioning.

Now, turn off the SEAL TEST switch.  The panel meter reads the output voltage of the
tracking circuit that, at this time, represents the command voltage necessary to keep the
whole-cell current zeroed.  This is, by definition, the resting voltage of the cell.  If you have
made the seal and gone whole cell in V-CLAMP mode, you will see a DC shift in the

Содержание Axopatch 200B

Страница 1: ...121 Rev D Printed in U S A Copyright 1997 1999 Axon Instruments Inc No part of this manual may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying microfilming recording or otherwise without written permission from Axon Instruments Inc QUESTIONS Call 650 571 9400 ...

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Страница 3: ... voltage 3 Temperature between 5 ºC and 40 ºC 4 Altitude up to 2000 m 5 This instrument is designed to be used under laboratory conditions Operate in a clean dry environment only Do not operate in a wet or damp environment Static Precautions The headstage can normally be safely handled However if you are in a laboratory where static is high i e you hear and feel crackles when you touch things you ...

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

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

Страница 6: ...xceder 10 del voltaje nominal 3 Temperatura entre 5 C y 40 C 4 Altitud hasta 2 000 m 5 Este instrumento está diseñado para ser usado en condiciones de laboratorio Debe operarse únicamente en un ambiente limpio y seco No lo use en un ambiente húmedo ni mojado Precauciones contra la estática El headstage puede manejarse con seguridad bajo condiciones normales Sinembargo si usted se encuentra en un l...

Страница 7: ...nt continu Gleichstrom Corriente continua Alternating current Courant alternatif Wechselstrom Corriente alterna On Supply Allumé alimentation An Netz Encendido suministro o Off Supply Éteint alimentation Aus Netz Apagado suministro On Supply Allumé alimentation An Netz Encendido suministro Off Supply Éteint alimentation Aus Netz Apagado suministro Protective conductor terminal Borne du conducteur ...

Страница 8: ...INFORMACION IMPORTANTE Axopatch 200B Copyright March 1997 1999 Axon Instruments Inc ...

Страница 9: ...N INSTRUMENTS INC VERIFICATION THIS INSTRUMENT IS EXTENSIVELY TESTED AND THOROUGHLY CALIBRATED BEFORE LEAVING THE FACTORY NEVERTHELESS RESEARCHERS SHOULD INDEPENDENTLY VERIFY THE BASIC ACCURACY OF THE CONTROLS USING RESISTOR CAPACITOR MODELS OF THEIR ELECTRODES AND CELL MEMBRANES DISCLAIMER THIS EQUIPMENT IS NOT INTENDED TO BE USED AND SHOULD NOT BE USED IN HUMAN EXPERIMENTATION OR APPLIED TO HUMA...

Страница 10: ...999 Axon Instruments Inc LATE INFORMATION Please check the back of this manual If there are pages in yellow these should be read first They contain errata and information that became available too late for inclusion in the body of the manual ...

Страница 11: ...2 Using the V CLAMP Mode 13 Adjustment of Pipette Capacitance Compensation 14 Adjustment of Leak Subtraction 14 Whole Cell Recording Model Cell 15 Pipette Offset Adjustment 16 Whole Cell Capacitance Compensation 16 Series Resistance Compensation 19 Brief Method For Setting Series Resistance Compensation 19 Detailed Method For Setting Series Resistance Compensation 22 Current Clamp Model Cell 28 Si...

Страница 12: ...ntials 50 Holding Command 50 Seal Test 51 Current Clamp 51 Membrane Potential 51 Whole Cell Current Clamp 51 Whole Cell Parameters 52 Electrode Capacitance Compensation 52 Limitations 53 Electrochemistry 54 Headstage 54 Offset Adjustment For Headstage 54 Frequency Boosting 55 Case Ground Connector 55 Mounting the Headstage 55 Cleaning 55 Static Precautions 56 Optical Pick up 56 Acoustic Pick up 56...

Страница 13: ... GENERAL INFORMATION 71 Grounding and Hum 71 Model Cell 72 Model Bilayer 73 Power Supply Glitches 74 Ten Turn Potentiometers 74 CHAPTER 8 REFERENCE SECTION PRINCIPLES OF OPERATION 75 Headstages 75 Principals of Operation 75 Resistor Feedback 75 Capacitor Feedback 76 If the Probe Output is Slow How Can Voltage Clamping Be Fast 78 What is Clamped During Voltage Clamping 79 Capacitance Compensation 7...

Страница 14: ...o Prevent Oscillations 90 Limitations 90 Pipette Offset 91 Leak Subtraction 92 Zap 93 Lipid Bilayers 93 Experimental Techniques 93 Noise vs Access Resistance 95 Current and Voltage Conventions 96 Trouble Shooting 101 Chapter 9 SPECIFICATIONS 103 Chapter 10 REFERENCES 119 Chapter 11 HEADSTAGE TUNING PROCEDURE 121 INDEX 127 WARRANTY 129 WARNING 131 CIRCUIT DIAGRAMS REQUEST 133 ...

Страница 15: ...tion detailed method 26 27 Figure 7 Pipette resistance measurement using SEAL TEST 32 Figure 8 Change in resistance while forming a seal 33 Figure 9 Going whole cell Capacity transients observed when rupturing the patch 37 Figure 10 Going whole cell Capacity transients observed during Amphotericin partitioning 38 Figure 11 Exploded view of the holder 59 Figure 12 Ag AgCl pellet assembly 60 Figure ...

Страница 16: ...charge the membrane capacitance 82 Figure 21 Integrator driving bilayer model 94 Figure 22 Typical current noise in bilayers as a function of series resistance 96 Figure 23 Total current noise as a function of bandwidth 105 106 Figure 24 Frequency tuning of whole cell configuration 123 Figure 25 Reset transient compensation 126 Table I Glass electrical and thermal properties 46 ...

Страница 17: ...Axopatch 200B achieves its ultra low noise performance by cooling components of the innovative capacitive feedback circuitry used for single channel recording This technology was developed by Axon Instruments engineers over a period of several years with extensive design and test contributions from scientific consultants The capacitive feedback technique is complicated The headstage output has to ...

Страница 18: ...time knowledgeable about the experimental techniques Therefore we have written this manual with two goals in mind The first is to explain the operation of the instrument and many of the principles that underlie its design The second is to provide the user with a guide to experimental techniques that our customers and scientific consultants have found useful during many years of experience in patch...

Страница 19: ...e factory Startup Procedure For the initial checkout the Axopatch 200B should be situated on a benchtop away from other equipment Do not install in a rack until the checkout is complete Make sure that the power is OFF An oscilloscope is the only other piece of equipment required for these tests A large sheet of aluminum foil is needed 1 The only connections to the Axopatch 200B should be a the pow...

Страница 20: ...T GAIN α 10 LOWPASS BESSEL FILTER 5 kHz LEAK SUBTRACTION MΩ OFF Test the noise 3 Shield the headstage with a large sheet of aluminum foil Wrap the foil loosely and completely around the headstage Leave room at the headstage input for the model cell in the next test Connect the foil shield to the ground input of the headstage gold plated 1 mm socket at the rear of the probe using a clip lead The ea...

Страница 21: ...e Also check on the screen of the oscilloscope for 60 Hz interference or other noise pickup Test the PIPETTE CAPACITANCE COMPENSATION and FILTERS 5 Set CONFIG to PATCH Set the oscilloscope gain to 0 5 V div trigger to line and the sweep speed to 2 ms div Connect the oscilloscope to the Scaled Output with a BNC cable Turn on the SEAL TEST switch You should see oppositely going capacitance transient...

Страница 22: ...ge the fit will be snug Make sure that the collet is inserted all the way so that the connector pin is fully inserted into the headstage input jack 8 Shield the headstage and model cell with the foil Reconnect the foil shield to ground 9 Place the CONFIG switch in the WHOLE CELL β 1 position Turn the panel meter switch to the I position Zero the panel meter using the PIPETTE OFFSET knob If the met...

Страница 23: ...V Turn the PIPETTE OFFSET control one full turn clockwise Meter should read 50 mV This is the voltage applied by the TRACK circuit to keep the current at zero 13 Turn the PIPETTE OFFSET control counterclockwise until the meter again reads 0 mV 14 Switch mode to I CLAMP NORMAL Switch meter to VHOLD IHOLD Rotate HOLDING COMMAND control until meter reads 1 0 nA Turn meter switch to Vm Meter should re...

Страница 24: ...ts get larger and faster Set the control to 100 Rotate the LAG control counterclockwise and verify that the circuit will oscillate at lower LAG values Test CAPACITANCE DITHER 19 Set up as in 15 and 16 above Connect square wave voltage source switching from approximately 0 V to 5 V at a frequency of between 0 5 to 5 Hz to CAP DITHER CONTROL BNC connection on back panel Set lowpass filter setting to...

Страница 25: ...EMP Verify that Probe Temp light is off and that temperature reading on Panel Meter is near 20 C note that reading is not strictly accurate at room temperature values Next set HEADSTAGE COOLING switch to ON Verify that Probe Temp light comes on when temperature reading falls below 0 C You have now verified that all the circuits are in working order ...

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Страница 27: ...NCE SECTION GENERAL INFORMATION An oscilloscope a step generator and some aluminum foil will be required Note that your headstage contains three feedback elements a capacitor in the PATCH position and resistors in the WHOLE CELL β 0 1 and β 1 positions On each setting the headstage gain is β mV pA and this gain is multiplied by the setting on the rotary gain switch α to achieve the total output ga...

Страница 28: ...tch to VTRACK MODE TRACK CONFIG PATCH OUTPUT GAIN α 10 LOWPASS BESSEL FILTER 5 kHz LEAK SUBTRACTION MΩ OFF Pipette Offset Adjustment Install the PATCH 1U model cell in BATH position into your headstage connector as described in the FUNCTIONAL CHECKOUT section Again surround it with aluminum foil grounded to the headstage ground connector Connect the SCALED OUTPUT BNC to one channel of your oscillo...

Страница 29: ...ipette offset voltage to achieve zero current This specifies the proper setting of the PIPETTE OFFSET control to zero junction potentials and electrode asymmetries Using the V CLAMP Mode Some investigators prefer not to use a tracking circuit but do their offset adjustments and sealing in V CLAMP The choice is a matter of personal preference Both methods enable you to follow drifts in the electrod...

Страница 30: ... over and over again until you find the setting where the transient is finally minimized Figure 2 With some practice you will be able to use the two controls simultaneously to compensate stray capacitance rapidly With real electrodes but probably not with your model cell the capacitance transient will have more than one component SLOW MAG and SLOW τ controls are provided for minimizing a second el...

Страница 31: ...ws PIPETTE OFFSET About 5 0 ZAP 0 5 ms SERIES RESISTANCE COMP PREDICTION 0 OFF SERIES RESISTANCE COMP CORRECTION 0 OFF SERIES RESISTANCE COMP LAG 1 µs WHOLE CELL CAP 0 pF OFF SERIES RESISTANCE 0 MΩ HOLDING COMMAND 0 mV toggle x1 OFF SEAL TEST OFF METER Set switch to VTRACK MODE TRACK CONFIG WHOLE CELL β 1 OUTPUT GAIN α 1 LOWPASS BESSEL FILTER 5 kHz LEAK SUBTRACTION MΩ OFF PULSE PULSE ON OFF 1 1 1 ...

Страница 32: ... the CELL position Insure that 60 Hz interference is sufficiently small If necessary this can be achieved by shielding with aluminum foil If necessary adjust PIPETTE OFFSET knob to zero the METER reading 3 For a Config setting of W Cell β 1 500 MΩ feedback resistor a gain setting of 0 5 is convenient for a Config setting of W Cell β 0 1 50 MΩ feedback resistor set the gain at 5 4 Begin with the PI...

Страница 33: ...ep speed e g 5 ms division and high vertical resolution e g 20 mV division adjust the LEAK SUBTRACTION potentiometer see Adjustment of Leak Subtraction above to eliminate leak current from the 500 MΩ resistor simulating the membrane resistance in the model cell as in Figure 3c 8 Turn the WHOLE CELL CAP switch on Reduce the vertical resolution to 1 V div and increase the sweep speed to 1 ms div Usi...

Страница 34: ...18 USE OF THE PATCH CLAMP A TUTORIAL Axopatch 200B Copyright 1997 1999 Axon Instruments Inc Insert figure 3 near here Figure 3 Whole cell capacitance compensation ...

Страница 35: ...roceed with the time constant RsCm of about 330 µs for the model cell where Rs is the series resistance and Cm is the membrane capacitance The Axopatch 200B uses dual controls to speed this response and to compensate for IR drops resulting from membrane current and series resistance and for the filtering effect of the membrane capacitance and series resistance These controls are labeled PREDICTION...

Страница 36: ...ESISTANCE control by about 2 3 As you reduce this setting the amplitude of the transient first decreases and then begins to increase A distinct minimum exists and the desired setting of the SERIES RESISTANCE control is at this minimum Figure 5c Next slightly decrease the FAST MAG setting The fast leading edge transient should decrease along with the peak to peak magnitude of the overall transient ...

Страница 37: ...USE OF THE PATCH CLAMP A TUTORIAL 21 Chapter 3 insert fig 5 near here Figure 5 Series resistance compensation brief method ...

Страница 38: ...P Figure 4 If the PREDICTION potentiometer has been advanced too far it may not be noticeable in 5 kHz bandwidth until the current begins to oscillate However in 100 kHz bandwidth you will observe ringing developing in the transient as the PREDICTION percentage becomes too large For best results you should start with whole cell transients canceled as in Figure 6a same as Figure 3d To begin advance...

Страница 39: ... Figure 6e Adjust the FAST τ of the PIPETTE CAPACITANCE COMPENSATION until the residual transient seems to be minimized Note that it will still be biphasic keep the waveform as smooth as possible The readjustment of FAST τ is usually not very large Now readjust the FAST MAG control of PIPETTE CAPACITANCE COMPENSATION and minimize the final transient A few iterative adjustments of the FAST MAG and ...

Страница 40: ...0 Hz The CORRECTION potentiometer is used to greatly reduce both of these errors To set CORRECTION properly first set the LAG potentiometer at about 10 µs Without changing any of the settings previously established advance the CORRECTION potentiometer gradually up to about 95 Observe the current at a vertical gain of about 100 mV div and a sweep speed of about 500 µs div As the CORRECTION percenta...

Страница 41: ...USE OF THE PATCH CLAMP A TUTORIAL 25 Chapter 3 insert fig 6 near here Figure 6 Series resistance compensation detailed method ...

Страница 42: ...26 USE OF THE PATCH CLAMP A TUTORIAL Axopatch 200B Copyright 1997 1999 Axon Instruments Inc insert fig 6 near here Figure 6 Series resistance compensation detailed method cont ...

Страница 43: ...positive feedback of the measured current Turning off this switch will result in a large ringing capacity transient with a peak to peak amplitude of more than 10 nA At a higher vertical gain on the oscilloscope e g 200 mV div 7 or 8 discernible peaks can be observed in this transient before they disappear into the noise about 2 ms following the beginning of the step command The membrane potential ...

Страница 44: ...l parameter measurements while in I CLAMP it is important to have the PIPETTE CAPACITANCE COMPENSATION set correctly before entering I CLAMP mode This is because in I CLAMP for a first approximation the pipette capacitance Cp appears to be in parallel with the membrane capacitance Cm If the PIPETTE CAPACITANCE COMPENSATION is set too low the measurement of Cm will be too high and vise versa Typica...

Страница 45: ...nge to a rise time of about 35 µs with less than 10 overshoot the internal rise time under these conditions is about 10 µs the signal at the I OUTPUT BNC is filtered at 10 kHz and thus is limited to a rise time of 35 µs SERIES RESISTANCE compensation is functional in I CLAMP mode when CORRECTION is on and may be used to compensate for the pipette resistance This is most important during intracellu...

Страница 46: ...RIES RESISTANCE COMP PREDICTION 0 OFF SERIES RESISTANCE COMP CORRECTION 0 OFF SERIES RESISTANCE COMP LAG 1 µs WHOLE CELL CAP 0 pF OFF SERIES RESISTANCE 0 MΩ HOLDING COMMAND 0 mV toggle x1 OFF SEAL TEST ON METER Set switch to VTRACK MODE TRACK CONFIG PATCH OUTPUT GAIN α 10 or as desired LOWPASS BESSEL FILTER 1 2 or 5 kHz LEAK SUBTRACTION MΩ OFF Set the oscilloscope to line triggering Switch off any...

Страница 47: ...des Drifting electrodes will cause a constantly changing VTRACK voltage in TRACK mode whereas in V CLAMP the current will continually drift off zero Switch the external command switch to the SEAL TEST position A 5 mV positive going rectangular pulse is applied to the patch clamp input The pulse frequency is the same as the AC line frequency and the duty cycle is 50 This will result in a square pul...

Страница 48: ...vestigators prefer to use mouth suction while others find that a 10 ml syringe works well With the latter approach it is best to start with the syringe plunger pulled back to a volume of 5 7 ml so that approximately this volume exists in the system before suction is applied Pulling the syringe back by about 1 ml from this starting volume will reduce the pressure by 0 2 to 0 14 atmospheres Since ma...

Страница 49: ...ecrease the time required to do the adjustments If a slow component remains minimize its size using the SLOW MAG and SLOW τ controls Turn off the SEAL TEST switch and you are ready to record channel currents In some experiments you may not require complicated pulse protocols and the application of a simple DC voltage to the Axopatch 200B command line is all that is required For this you can use th...

Страница 50: ...ange of some converters The Axopatch 200B includes a leak subtraction circuit to alleviate this problem With the HOLDING COMMAND applied turn the LEAK SUBTRACTION control until the baseline current returns to zero If the baseline current is really seal current it should exhibit linear behavior Once the LEAK SUBTRACTION is set properly it should be possible to toggle the HOLDING COMMAND switch betw...

Страница 51: ...n that is low in Ca2 and the tip should be as large as possible to minimize the pipette resistance the importance of low pipette resistance is obvious from the previous series resistance compensation discussion At this point proceed to insert the holder lower the pipette into the bath and make a seal in the same manner as described in the preceding single channel recording tutorial A pulse of stro...

Страница 52: ... the application of moderate suction while the voltage pulse is given results in a higher incidence of successful patch disruption The reappearance of the original rectangular pulse either means that you have lost the seal or that the cell does not have a large input resistance It is not unusual for small cells to have an input resistance of several gigohms but with active conductances it might be...

Страница 53: ...tents are not washed out as in standard whole cell techniques This is an advantage or a disadvantage depending on the experiment With this technique a rise in whole cell capacity transients will be observed as the antibiotic partitions into the cell as shown in Figure 10 400 pA 3 ms 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN Figure 10 Going whole cell capacity transients observed during amphortericin partition...

Страница 54: ...If there is still a DC current shift i e their anticipated voltage was not quite correct minor adjustments of the HOLDING COMMAND control can be made to zero the current Another possible scheme to go whole cell without loss of resting potential is to disrupt the patch while in I CLAMP mode The current clamp circuit is fast enough to keep the membrane current at zero and thus keep the cell at its r...

Страница 55: ...sed in the Whole Cell Recording Model Cell section above The Axopatch 200B contains a 4 pole internal Bessel filter with five frequencies for filtering the current output For many experiments the 1 2 5 10 and 100 kHz settings available will be sufficient If other frequency cutoffs are required they must be provided from an external filter Again be sure that you take into account that the final ban...

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Страница 57: ...AND because it is often useful to be able to disconnect the computer input via the front panel The scaled and filtered current is also available either from a front panel or rear panel BNC SCALED OUTPUT One channel of the computer s A D converter can be connected to this output for simultaneously sampling the current as the computer is delivering command voltages through its D A converters The I O...

Страница 58: ...er or a digital output to activate this line either just before or simultaneously with the application of a voltage step to the EXT COMMAND input The duration of the blanking input would be that expected for the duration of the capacitance transient Two rear panel BNCs are provided specifically for use in single channel recording with the integrating headstage One is the FORCED RESET INPUT A disad...

Страница 59: ... any particular noise source that is large will dominate the total noise and make other noise sources insignificant Therefore all potentially contributing noise sources must be minimized Specifically the headstage the pipette glass the holder and the seal contribute significantly even under circumstances where extraneous noise pickup from the environ ment is negligible It is absolutely crucial tha...

Страница 60: ...Tanaka Biophysical J 53 287 292 1988 Since any glass may potentially modify channel currents one must be aware of this fact and control for it regardless of the glass one uses We recommend two glasses Corning 7052 and quartz Both have been successfully sealed to many different cell types Quartz with its significantly lower loss factor has produced the lowest noise recordings known to us However be...

Страница 61: ... eliminated While many other hydrophobic substances have been used none to the best of our knowledge produces as low noise as does Sylgard 184 Sylgard also decreases the capacitance of the pipette wall and so reduces the lossiness of the wall as well It has been shown experimentally that Sylgard will improve the noise of any glass but it will not turn a poor electrical glass into a good one Low lo...

Страница 62: ...120 80 10 1 6 7 630 High lead EG 16 90 11 3 9 6 580 High lead 7040 1 00 9 6 4 8 700 Kovar seal borosilicate KG 12 1 00 9 9 6 7 632 High lead 1723 1 00 13 5 6 3 910 Aluminosilicate 0010 1 07 8 9 6 7 625 High lead 7052 1 30 9 2 4 9 710 Kovar seal borosilicate EN 1 1 30 9 0 5 1 716 Kovar seal borosilicate 7720 1 30 8 8 4 7 755 Tungsten seal borosilicate 7056 1 50 10 2 5 7 720 Kovar seal borosilicate ...

Страница 63: ...hecked before each attempt to make a seal When the holder with a filled pipette has been inserted into the headstage connector and the pipette tip is positioned just above the bathing solution the rms current noise seen on the meter of the Axopatch 200B meter switch in the IRMS position should not significantly exceed 0 08 pA Seal The seal will usually be the dominant noise source if it is only a ...

Страница 64: ...ise source to consider is the noise in the signal generator that provides the command In the Axopatch 200B we have succeeded in minimizing this noise by heavily attenuating the external command However it is possible for this noise source to be significant particularly if the command signal comes from a D A converter ...

Страница 65: ...rately reflect the cell membrane potential Changes in resting potential spike activity and synaptic activity may be monitored in this way however please note risetime limitations listed in the SPECIFICATIONS chapter for current clamp Compensating for electrode resistance in current clamp is straightforward Monitor the voltage with SEAL TEST activated or when applying a step current command you wil...

Страница 66: ...nd that increases to 500 kΩ resistor while a TTL signal is high Command Potentials Command potentials can be obtained from two internal sources HOLDING COMMAND and SEAL TEST and from external sources via two rear panel BNCs Holding Command In voltage clamp this control allows the user to apply a membrane holding potential of 200 mV with the toggle set to x1 the full range of 1V is available when t...

Страница 67: ...an be improved by using the FAST and SLOW ELECTRODE CAPACITANCE COMPENSATION as well as the SERIES RESISTANCE COMPENSATION controls Membrane Potential In voltage clamp mode Vm is simply equal to the command potential In current clamp mode Vm is not clamped and automatically becomes the voltage needed to allow the commanded current to flow Note To avoid introducing errors you should not change the ...

Страница 68: ...or values Whole Cell Parameters Although WHOLE CELL CAP is disabled in current clamp mode SERIES RESISTANCE compensation is active and may be used to correct for pipette resistance Thus one may use the Axopatch 200B to accurately record synaptic action and field potentials from cells and tissues as well as to record currents from patches and cells Please note however the risetime limitations liste...

Страница 69: ...pipette the voltage gain of the headstage is nearly equal the value of the feedback resistor divided by the value of the pipette resistance In the WHOLE CELL mode and with a pipette resistance of 1 MΩ this voltage gain is 500 50 for β 0 1 In order to guarantee stability with pipette resistances as low as 1 MΩ the current clamp circuitry must be deliberately slowed down compromising the response ti...

Страница 70: ...rovements over previous headstages The overall size is smaller and narrower than other headstages allowing for greater ease of placement during experiments Three configuration choices one patch and two whole cell provide a wide useful range of operation Most importantly due to improved circuitry and temperature control provided by an internal Peltier cooling unit the CV 203BU offers the lowest sin...

Страница 71: ...ensions The headstage box measures 0 7 wide x 0 75 high x 4 2 long Mounting the Headstage The improved slim design of the CV 203BU headstage makes it easier to guide your pipette towards your preparation without physical interference from other instruments in your set up For maximum mechanical rigidity the headstage can be mounted directly to some manipulators using the mounting plate located on t...

Страница 72: ...gth to activate the input transistors inside the hybrid Therefore you should prevent bright light from falling on the input connector with a shade or by dimming lights If you notice line frequency hum on the current record it could be due to fluctuating light levels from a bright fluorescent light or equivalent In general low light levels are not a problem Acoustic Pick up Rare instances have been...

Страница 73: ...er can be fully disassembled for cleaning and parts replacement Mechanical stability of the pipette is assured in several ways For example as the pipette cap is closed the cone washer is compressed on the pipette from the force applied to the front and back of the cone washer The holder mates with the special threaded Teflon connector on U type Axon Instruments headstages and is secured in place w...

Страница 74: ...th a large ID i e 1 1 mm Therefore the HL U is supplied with 0 25 mm silver wire Spare components included with each holder are as follows one 50 mm length of silver wire 40 cone washers 10 of each size and one 70 mm length of silicone tubing Cut into 2 mm lengths the silicone tubing will yield approximately 30 replacement silicone seals Additional pipette caps cone washers silicone tubing pins an...

Страница 75: ... by the sharp back end of the pipette you can smooth the pipette edges by rotating the back end of the pipette in a bunsen burner flame before filling the pipette with salt solution Cleaning For lowest noise keep the holder clean Frequently rinse the holder with distilled water If more thorough cleaning is required briefly wash in ethanol or mild soapy water Never use methanol or strong solvents F...

Страница 76: ... end of the pipette extends the life of the chloride coating by minimizing the amount of scratch damage Another way to protect the AgCl coating is to slip a perforated Teflon tube over the chlorided region The chlorided region should be long enough so that the pipette solution does not come in contact with the bare silver wire Replacing the Silver Wire To replace the silver wire insert the nonchlo...

Страница 77: ...ntional BNC type holders to be used with Axon Instruments U type headstages Use the HLB U with all U type CV and HS headstages e g CV 4 1 100U and HS 2A x1MGU These headstages have a threaded white Teflon collet Input BNCs BLANK ACTIVATE INPUT A positive TTL pulse delivered to this input causes the output current to be held at the value it had at the instant the pulse was activated and to hold it ...

Страница 78: ...command inputs are summed together inside the patch clamp FORCED RESET INPUT Causes the integrator and differentiator to reset on the positive edge of a TTL pulse Used for example to force a reset just before applying a command to a cell or membrane patch This increases the probability that no reset will occur during the brief recording period following the command SPEED TEST INPUT When switched o...

Страница 79: ...instead of its amplitude response or based on a straight line approximation to the filter characteristics instead of the actual characteristics You should check your external filter by checking tr for a step signal applied to its input When a signal with 10 90 rise time t1 is passed through a filter with 10 90 rise time t2 the rise time of the output signal is approximately Output Gain α α α α The...

Страница 80: ...ic range the Axopatch 200B uses a 500 MΩ feedback resistor The output of the current to voltage converter is therefore 0 5 mV pA To simplify the daily mental scaling task for the user the headstage output is presented to the user as 1 mV pA by including an additional two times x2 gain In PATCH mode the output of the differentiator is also 0 5 mV pA and therefore similar considerations apply Output...

Страница 81: ...mputer to determine the setting of the gain switch The gain takes α and β factors into account I mV pA 0 051 0 11 0 21 0 5 1 2 5 10 20 50 1002 2002 5002 Vm mV mV N A N A N A 0 5 1 2 5 10 20 50 100 200 500 Telegraph Output V 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 These values have been chosen to fit in the range 0 10 V so that they can be read on A D converter channels of most computer...

Страница 82: ...front panel two LEDs specify whether the output is current I or membrane voltage Vm The current gain is α βmV pA The voltage gain is α mV mV 10 Vm OUTPUT Provides the membrane voltage multiplied by ten x10 for use in current clamp experiments In V CLAMP mode 10 Vm is ten times the command potential If the pipette current is zero or if the series resistance correction is 100 the command potential i...

Страница 83: ...e set on the HOLDING COMMAND potentiometer When VHOLD IHOLD is selected it will show the absolute value of the HOLDING COMMAND even if the HOLDING COMMAND control is OFF 2 I The pipette current The reading is automatically scaled to suit the headstage gain The operation is autoranging that is the decimal point and the units indicators automatically shift so that large DC currents can be displayed ...

Страница 84: ...rd 2 Use a screwdriver or a similar device to rotate the fuse holder counterclockwise 3 Replace the fuse with another fuse of the same rating 4 Reconnect the power cord Probe Temperature The green HEADSTAGE COOLED light comes on when the temperature of the Peltier device that cools the critical components of the headstage drops below 0 C This light should go on very soon after power is switched on...

Страница 85: ...olarizing voltage 1 3 VDC to the patch for a controlled duration This often causes dielectric breakdown of the membrane Suggested Use Apply a repetitive test command e g Seal Test Start with Duration 0 5 ms Press Trigger to ZAP the membrane Successful zapping is accompanied by an increase in the current noise and by large capacitance charging current transients in response to the test command Use ...

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Страница 87: ...tion bath only by directly connecting it to the gold ground connector on the back of the headstage 2 Place the Axopatch 200B in the rack in a position where it will not absorb radiation from adjacent equipment A grounded thick sheet of steel placed between the Axopatch 200B and the radiating equipment can effectively reduce induced hum 3 Initially make only one connection to the Axopatch 200B from...

Страница 88: ...n Model Cell The PATCH 1U model cell Figure 13 can be used to assist with testing and setting up The pipette is modeled by a 10 MΩ resistor the cell is modeled by 500 MΩ in parallel with 33 pF the membrane time constant is 16 5 ms and the patch is modeled by a 10 GΩ resistor The pipette capacitance is about 4 6 pF The charging time constant is approximately 330 µs 10 MΩ x 33 pF The PATCH 1U model ...

Страница 89: ...0G BATH ACTUAL CIRCUIT 10M Ω Ω Ω Ω Ω Ω Ω Figure 13 PATCH 1U model cell Model Bilayer The MCB 1U model bilayer contains a 10 kΩ resistor that models the pipette in series with a 100 pF capacitor that models the bilayer membrane Figure 14 EQUIVALENT CIRCUIT 10 kΩ ACTUAL CIRCUIT 100 pF BILAYER 10 kΩ 100 pF BILAYER 4 pF Figure 14 MCB 1U bilayer model ...

Страница 90: ...ng made 2 Water baths heaters coolers etc should operate from zero crossing relays 3 RFI1 filters should be installed in glitch producing equipment In most circumstances occasional transients on the outputs are inconsequential and therefore no precautions have to be taken Ten Turn Potentiometers The ten turn potentiometers used in the Axopatch 200B are high quality wirewound types An inherent prob...

Страница 91: ...s the voltage output is proportional to the current input In contrast conventional microelectrode amplifier headstages are voltage followers in which the voltage output corresponds to the voltage input Resistor Feedback The essential parts of a resistive headstage are shown in Figure 15 Vp BOOST R I f OFFSET AND SCALING HIGH FREQUENCY BOOST I Vo CIRCUIT f PROBE Figure 15 Resistive headstage ...

Страница 92: ...d for patch clamping is that the output bandwidth of the probe is inherently low To a first approximation the bandwidth is set by the product of Rf and the stray capacitance across it For example if Rf is 500 MΩ and the stray capacitance is 0 5 pF the bandwidth is about 600 Hz To overcome this limitation the probe output is passed through a high frequency boost circuit The gain of this circuit is ...

Страница 93: ...y noise can be substantially less than that of the 50 GΩ feedback resistor customarily used in resistive headstages At the same time the high frequency noise is less because the capacitor lacks the excess high frequency noise associated with gigohm value resistors While the integrating headstage is quieter and more linear than resistive feedback headstages there is one disadvantage The voltage acr...

Страница 94: ...k elements The capacitive element is selected using the front panel CONFIG switch in the PATCH position while the resistive headstage is engaged when the switch is in the WHOLE CELL position The capacitive feedback PATCH is recommended for single channel recording because it offers the lowest noise In such recordings the average current is not more than a few pA and so the resets are infrequent In...

Страница 95: ...ming junction is fast In this discussion we have carefully referred to the fact that it is the pipette that is rapidly voltage clamped The membrane potential is voltage clamped to its final value much more slowly To a reasonable approximation the time constant for voltage clamping the membrane is RpCm where Rp is the pipette resistance and Cm is the membrane capacitance What is Clamped During Volt...

Страница 96: ...e transient signal on the output I By properly setting the fast and slow magnitude and τ controls a current IC1 can be induced in capacitor C1 connected to the headstage input to exactly equal Ip In this case no current needs be supplied by Rf and there is no transient on the output The FAST controls compensate that part of Cp that can be represented by a lumped capacitance at the headstage input ...

Страница 97: ...rrent injected through C2 will supply the transient membrane current Im These adjustments do not alter the time constant for charging the membrane Their function is to offload the burden of this task from the feedback resistor Rf In many cells even a small command voltage Vc of a few tens of millivolts can require such a large current to charge the membrane that it cannot be supplied by Rf The hea...

Страница 98: ... n A A A Af f f ft t t te e e er r r r W W W Wh h h ho o o ol ll le e e e c c c ce e e el ll ll ll l C C C Co o o om m m mp p p pe e e en n n ns s s sa a a at t t ti ii io o o on n n n VC m V I Rf C2 I Figure 20 Using the injection capacitor to charge the membrane capacitance After perfect whole cell compensation is applied the current to charge the membrane capacitor is removed from the IRf trace...

Страница 99: ...ignificantly greater than Rs Series Resistance In patch clamping the term refers to the pipette resistance plus any other access resistances to the patch or cell The SERIES RESISTANCE and WHOLE CELL CAP controls along with PREDICTION and CORRECTION are active in WHOLE CELL mode In PATCH mode only SERIES RESISTANCE and CORRECTION are functional Absolute Value The absolute range of the series resist...

Страница 100: ...ithin 1 of its final value will require nearly 10 ms after the start of a step command Note that the uncompensated whole cell capacity transient has the shape of the derivative of the true membrane potential and both will have the same time constant 2 Uncompensated series resistance will also cause the membrane potential to deviate from the command potential when ionic membrane current Im flows Th...

Страница 101: ...ting of the WHOLE CELL CAP control and a time constant that is determined by the setting of both the WHOLE CELL CAP and SERIES RESISTANCE controls Precise canceling the whole cell capacity transient with these controls requires a unique setting in each case These settings are accurate representations of Rs and Cm to within 2 3 As will be described below the use of PREDICTION will modify the time c...

Страница 102: ...l is set at 50 pF so that the whole cell capacity transient is perfectly canceled If the PREDICTION control is OFF 0 the signal applied to the headstage 5 pF capacitor 50 pF for β 0 1 in response to a step voltage command will have a time constant of 500 µs and an amplitude that is appropriate to cancel a whole cell capacitance transient arising from these parameters about 10 Vc With 0 PREDICTION ...

Страница 103: ...at the headstage by a 10 1 voltage divider Since the circuitry in the Axopatch 200B mainframe will saturate at about 11 12 V Vcp is limited in absolute value to about 1 1 to 1 2 V To be conservative we will use 1 1 V in the following calculations The peak amplitude of Vcp for a step voltage command Vc is given by Vc Rs Rsrp which can be rewritten as Vc 1 PREDICTION 100 So we may state the limitati...

Страница 104: ...e TUTORIAL It should be noted that PREDICTION will work for any command waveform not just steps This may be useful for capacitance measurements using phase sensitive techniques or lock in amplifiers Although PREDICTION can greatly speed the response time of the true membrane potential with respect to the command potential and thus overcome one important effect of series resistance it does not corr...

Страница 105: ...width for current measurement is increased to 3 2 kHz in this example With 95 CORRECTION the possible bandwidth is increased to 6 4 kHz and with 98 it is further increased to 16 kHz although the effects of LAG should not be forgotten If the capacity transient has been canceled prior to the use of CORRECTION and for now assume that PREDICTION has already been set at 95 then in principle there is no...

Страница 106: ...h the stray capacitance of the pipette into the bath The current that flows through the pipette resistance into the cell is the current that is intended to be compensated The CORRECTION circuitry also tries to compensate for the current into the pipette capacitance However in this case there is no significant series resistance component to compensate and the CORRECTION circuit will oscillate as so...

Страница 107: ...al The effect will be a larger transient at voltage levels that activate the fall of membrane resistance If the cell input resistance becomes comparable to or less than the pipette resistance the whole cell patch technique will probably not work Note that typical patch pipette resistances are in the range of 2 to 10 MΩ In this case it would be preferable to use a discontinuous chopped single elect...

Страница 108: ...0 Leak Subtraction The passive membrane response to a voltage step consists typically of a transient and a steady state component It is often helpful to subtract these from the output so that only active responses are observed The transient component is eliminated by using the CAPACITANCE COMPENSATION controls as discussed in the Capacitance Compensation section above The steady state component is...

Страница 109: ... pF one has the ability of minimizing noise by minimizing access resistance To realize the full potential of the design the user should externally initiate a reset at each voltage step applied to the membrane This is accomplished by applying a positive going TTL pulse to the FORCED RESET INPUT BNC on the rear panel Because of internal timing delays the positive going edge of the reset pulse can oc...

Страница 110: ...reset of the integrator is initiated internally an external reset pulse is not needed In the reset state switch S1 is closed and the 1 pF capacitor is shunted by the 10 kΩ reset resistor This all happens within 2 µs from the time the integrator reaches 10 V or 10 V During the time the integrator is in reset 10 µs it can pass up to 1 mA 106 nA of current to charge the membrane For example if Cm 100...

Страница 111: ...nce the lower the noise While there will be some lower limit on the value of the access resistance it will not be set by stability criteria of the instrument In bilayer applications one is typically working with bandwidths below 1 kHz In this region the enCin noise has not yet become the major contributor to the overall noise where en is the voltage noise of the probe input FETs1 and Cin is the ca...

Страница 112: ...n this discussion applies to all amplifiers manufactured by Axon Instruments Positive Current The flow of positive ions out of the headstage into the microelectrode and out of the microelectrode tip into the preparation is termed positive current Inward Current Current that flows across the membrane from the outside surface to the inside surface is termed inward current Outward Current Current tha...

Страница 113: ...polarizing voltage across the membrane This convention is adhered to even if the current is so large that the absolute magnitude of Vm becomes larger For example a current that causes Vm to shift from 70 mV to 90 mV is still said to depolarize the cell Stated simply depolarization is a positive shift in Vm Conversely hyperpolarization is a negative shift in Vm Whole Cell Voltage and Current Clamp ...

Страница 114: ...nvestigators chose to call the negative current that they measured a depolarizing current because it corresponded to the depolarizing sodium current This choice while based on sound logic was unfortunate because it means that from the recording instrument s point of view a negative current is hyperpolarizing in intracellular current clamp experiments but depolarizing in voltage clamp experiments T...

Страница 115: ...xamples it can be seen that the transmembrane patch potential varies inversely with changes in the command potential shifted by the resting membrane potential RMP of the cell A positive pipette current flows through the pipette across the patch membrane into the cell Therefore a positive current is inward Inside Out Patch In this mode of recording the membrane patch is detached from the cell with ...

Страница 116: ...ne conditions assuming that Vm 70 mV the potential inside the pipette must be adjusted to 70 mV If the potential inside the pipette is then increased from 70 mV to 50 mV the transmembrane potential of the patch depolarizes from 70 mV to 50 mV The membrane potential changes directly with the command potential A positive pipette current flows through the pipette across the patch membrane from the in...

Страница 117: ...y equipment connected to our instruments If you have a problem please disconnect all instruments connected to the Axopatch 200B except for the headstage Ideally remove the Axopatch 200B from the rack Work completely through the Functional Checkout This can often uncover a problem that is in your set up If the problem persists please call us for assistance Another common problem is caused when dirt...

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Страница 119: ...ion All critical components are in a sealed hybrid Configuration High speed low noise current to voltage converter Headstage Gain β β β β 1 mV pA in either PATCH or WHOLE CELL β 1 mode 0 1 mV pA in WHOLE CELL β 0 1 mode Feedback Element PATCH 1pF WHOLE CELL β 1 500 MΩ in parallel with 1pF WHOLE CELL β 0 1 50 MΩ in parallel with 1pF ...

Страница 120: ...ntained in the main instrument Tuning is automatically bypassed when the capacitive feedback is selected Pipette Capacitance Compensation Injection Capacitor Value 1 pF Whole Cell Capacitance Compensation Injection Capacitor Values PATCH mode none WHOLE CELL mode β 1 5 pF β 0 1 50 pF Case Case connected to ground Case jack mates to 2 mm plugs Bandwidth Test signal applied via SPEED TEST input PATC...

Страница 121: ... 005 pAp p 0 005 pAp p 0 005 pAp p 0 1 100 Hz 0 030 pAp p 0 50 pAp p 1 60 pAp p 0 1 1 kHz 0 015 pA rms 0 25 pA rms 0 75 pA rms 0 1 5 kHz 0 060 pA rms 0 65 pA rms 1 65 pA rms 0 1 10 kHz 0 130 pA rms 1 10 pA rms 3 00 pA rms With holder 0 1 10 kHz 0 145 pA rms 1 10 pA rms 3 00 pA rms 2 2 10 29 30 10 10 Hz 100 Hz FREQUENCY 1 kHz 700 GΩ CURRENT NOISE DENSITY A Hz 10 31 32 10 Figure 23A Typical low freq...

Страница 122: ...OISE DENSITY A Hz 10 29 10 10 10 30 31 32 10 Hz 100 Hz 1 kHz 10 kHz 2 FREQUENCY Figure 23B Typical broadband current noise spectrum patch mode 0 1 1 0 01 0 001 100 Hz 1 kHz 10 kHz BANDWIDTH TOTAL NOISE pA RMS Figure 23C Typical total current noise as a function of bandwidth patch mode ...

Страница 123: ... cause a reset 12 pC of charge needed whereas a 40 mV step will not 8 pC of charge needed Reset transients in current waveform at Scaled Output typical 100 Hz bandwidth 0 25pA 1 kHz 0 5 pA 10 kHz 2 pA Current Clamp The current clamp mode has two speed settings I CLAMP NORMAL and I CLAMP FAST I CLAMP NORMAL is for use with electrode resistances greater than 1 MΩ I CLAMP FAST is for use with pipette...

Страница 124: ... 1 MΩ 0 MΩ 0 pF 15 µs 10 N A 1 MΩ 500 MΩ 33 pF 350 µs 0 N A 10 MΩ 0 MΩ 0 pF 200 µs 20 20 µs 1 10 MΩ 500 MΩ 33 pF 250 µs 10 10 µs 1 50 MΩ 500 MΩ 33 pF 500 µs 30 150 µs 1 HL U Pipette Holder HL U holder mates to threaded Teflon input connector of the CV headstage Post for suction tubing is 1 mm OD HL U holder accepts glass 1 0 1 7 mm OD Supplied with silver wire Optional HLR U right angle adapter an...

Страница 125: ...al to setting on CELL CAPACITANCE TELEGRAPH OUTPUT The range of values for the SERIES RESISTANCE compensation control is 0 100 MΩ Series Resistance Compensation Prediction OFF 0 100 Acts with WHOLE CELL PARAMETERS to speed up charging of the membrane Maximum achievable PREDICTION is limited by magnitude of voltage step see Figure 4 Correction OFF 0 100 Acts with SERIES RESISTANCE setting to reduce...

Страница 126: ...educing the Whole Cell Capacitance value by that amount Mode V Clamp Pipette voltage is clamped I Clamp Normal or Fast Pipette current is clamped to command current from Holding Command knob or external input NORMAL mode stable for electrode resistances greater than 1 MΩ FAST mode stable for electrode resistances greater than 10 MΩ SERIES RESISTANCE control is active I 0 Slow I CLAMP to zero curre...

Страница 127: ...eal Test 5 mV command at line frequency voltage clamp mode 50 pA current clamp mode β 1 or 500 pA current clamp β 0 1 External Commands Two separate BNC inputs one front switched one rear switched Sensitivity FRONT SWITCHED 20 mV V in V CLAMP 2 β nA V in I CLAMP disabled in TRACK and I 0 REAR SWITCHED 100 mV V in V CLAMP 2 β nA V in I CLAMP disabled in TRACK and I 0 Input impedance 10 kΩ Inputs ma...

Страница 128: ...nA for β 1 100 nA for β 0 1 toggle x5 Disabled in TRACK and I 0 mode Pipette Offset Manual 250 mV Ten turn control with uncalibrated dial Track I 0 200 mV Nulling potential automatically adjusts to main tain zero pipette current RMS Noise 3 5 digit meter displays rms current noise in pA Measurement bandwidth is 30 Hz to 5 kHz Upper 3 dB frequency is set by 4 pole Butterworth filter Inputs Forced R...

Страница 129: ... Outputs Scaled Output Scaled and filtered by output control settings Sample and hold pedestal compensation Output is I αβ mV pA when in V CLAMP or TRACK I 0 mode Output is Vm α mV mV when in I CLAMP mode BNCs on front and rear panels are identical I Pipette current Rear switched gain of either βmV pA or 100 βmV pA fixed filter 10 kHz 3 pole Bessel Output does not benefit from sample and hold pede...

Страница 130: ...rom current record Range 100 βMΩ to Telegraph Outputs Gain Takes α and β gain factors into account I mV pA 0 051 0 11 0 21 0 5 1 2 5 10 20 50 1002 2002 5002 Vm mV mV N A N A N A 0 5 1 2 5 10 20 50 100 200 500 Telegraph Output V 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 Frequency Filter Setting kHz 1 2 5 10 100 Telegraph Output V 2 4 6 8 10 Mode I CLAMP I CLAMP Mode TRACK V CLAMP I 0 NORM...

Страница 131: ...splays TRACK potential VTRACK in mV membrane potential Vm in mV current noise IRMS in pA rms membrane current I in pA or nA HOLDING COMMAND VHOLD IHOLD in mV or nA or capacitor feedback circuitry temperature in degrees Celsius TEMP Meter has autoranging feature when membrane current I or holding command VHOLD IHOLD is chosen Grounding Signal ground is isolated from chassis and power ground Signal ...

Страница 132: ...layer Emulates a bilayer membrane 10 kΩ resistor in series with a 100 pF capacitor Headstage Dimensions Case is 0 7 x 0 75 x 4 2 17 8 x 19 x 106 7 mm A removable mounting plate 2 5 x 2 0 x 0 25 is supplied Cable length is 10 feet 3 m Cabinet Dimensions 3 5 89 mm high 19 483 mm wide 12 5 317 mm deep Mounts in standard 19 rack Handles included Net weight 11 5 lbs 5 1 kg Supply Requirements Line Volt...

Страница 133: ...SPECIFICATIONS 117 Chapter 9 Accessories Provided Theory and Operation Manual HL U Pipette Holder Spare fuse PATCH 1U Model Cell MCB 1U Model Bilayer DR 1 Series Resistance Dither Unit ...

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Страница 135: ...derland MA Sinauer Associates Inc 1992 Kawagoe K T Zimmerman J B and Wightman R M Principles of voltammetry and microelectrode surface states Journal of Neuroscience Methods 48 225 240 1993 Joshi C and Fernandez J M Capacitance Measurements An analysis of the Phase Detector Technique Used to Study Exocytosis and Endocytosis Biophysical Journal 53 885 892 1988 Purves R D Microelectrode Methods for ...

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Страница 137: ... p square wave into the headstage input 3 Has determined that the reset transients in PATCH mode are more than twice the typical values see SPECIFICATIONS This should be verified by warming up the unit for at least one hour and connecting the PATCH 1U model cell PATCH position as a load Set the HOLDING COMMAND to or 200 mV to induce resets toggle set to x1 Monitor the SCALED OUTPUT BNC and trigger...

Страница 138: ... METER Use JUNCTION NULL to zero the current Set HOLDING COMMAND to 100 mV toggle set to x1 Switch HOLDING COMMAND between and Trim RT4 so that the difference on the meter is 20 nA nominally 10 nA if the current has been exactly zeroed Repeat procedure with CONFIG set to WHOLE CELL β 1 and trim RT5 Repeat procedure with CONFIG set to WHOLE CELL β 0 1 and trim RT33 Remove PATCH 1U model cell Use ap...

Страница 139: ...div 1 ms div Apply a 10 Vp p 100 Hz triangle wave to the SPEED TEST BNC On the scope you should see about a 1 Vp p square wave If the square wave response is unacceptable in terms of rise time or linearity continue with procedure otherwise move on to RESET TRANSIENT COMPENSATION Set FILTER to 10 kHz Adjust RT31 until you get a response which looks like either trace a of Figure 24 Figure 24 Frequen...

Страница 140: ...ure The results rely on the patience and skill of the person who is doing the adjustments Please confirm that the reset transients are indeed unacceptable before proceeding Because of the consistency between units you may not have to do these adjustments even if you are changing headstages Make sure the unit has been on continually for at least one hour Forced Reset Remove PATCH 1U model cell CONF...

Страница 141: ...out twice a second at either or 200 mV If there is an obvious time difference between and trim RT24 CHG INJ to balance it out Set switches 3 4 and 5 of SW8 to OFF internal reset compensation Find reset transient on scope It should have about a 2 7 pA amplitude with an apparent decay time constant of roughly 0 5 ms see Figure 25b The transient should be riding on a DC level of or 20 pA and should c...

Страница 142: ...mains it can be compensated with RT25 DIAB 3 MAG and RT26 DIAB 3 TAU For the most accurate adjustment of these trims a 100 Hz 4 pole or greater Bessel filter should be connected between the SCALED OUTPUT and the scope The next steps assume the 100 Hz filter is installed Set scope to 20 mV div 50 ms div Set switch 5 of SW8 to ON Trim RT25 and RT26 to minimize slowest transient As above some re trim...

Страница 143: ...n 51 Initiation 51 Model Cell 28 Speed 53 Whole cell 51 Current Convention 96 Current Output 68 Current Display 67 Data Not Valid Output 64 Electrochemistry 54 External Command 62 72 Filter See Output 63 Forced Reset 62 Frequency Telegraph 64 Fuse Changing 68 Gain See Output 63 Gain Telegraph 64 Glass Dimensions 61 Types 46 Ground Connections 4 6 55 Grounding 71 Headstage Adapters 61 Capacitor Fee...

Страница 144: ...Meter 67 Percentage Compensation See Series Resistance 83 Perforated Patch 36 Pipette 55 Chloriding 60 Cleaning 59 Filling 59 Glass 46 Insertion 59 Nulling 67 Offset 12 91 potentiometers 74 Power Supply Glitches 74 Voltage Selection 68 Prediction 83 Reset 77 Forced Reset 62 RMS Noise Display 5 67 Scaled Output 66 Seal Test 5 51 Series Resistance Compensation 19 83 Correction 19 83 Fast 19 Lag 19 8...

Страница 145: ...ng of the product to the customer Before returning products to our factory the customer must contact us to obtain a Return Merchandise Authorization number RMA and shipping instructions Failure to do so will cause long delays and additional expense to customer This warranty shall not apply to damage resulting from improper use improper care improper modification connection to incompatible equipmen...

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Страница 147: ...RAGILE and an arrow showing which way is up We do not recommend using loose foam pellets to protect the Axopatch 200B If the carton is dropped by the shipper there is a good chance that the Axopatch 200B will shift within the loose pellet packaging and be damaged If you need to ship your Axopatch 200B to another location or back to the factory and you do not have a means to adequately package it A...

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Страница 149: ...ne orders will not be accepted Name of registered owner __________________________________________________________________ Department _____________________________________________________________________________ University Institute _______________________________________________________________________ Street address ___________________________________________________________________________ City...

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Страница 151: ...CIRCUIT DIAGRAMS REQUEST FORM 135 Circuit Diagrams Request Form Please fold out so that you may refer to this page while reading the manual ...

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Страница 153: ...Circuit Diagrams Request Form ...

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