1. Manuals
  2. Brands
  3. Axon Manuals
  4. Industrial Equipment
  5. Axopatch 200B
  6. Theory of operation

Axon Axopatch 200B Theory Of Operation

Patch clamp
Hide thumbs
Table of Contents

Advertisement

Quick Links

March 1999
Axopatch 200B
Patch Clamp
Theory and Operation
Part Number 2500-121 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
Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

Advertisement

Table of Contents
loading
Need help?

Need help?

Do you have a question about the Axopatch 200B and is the answer not in the manual?

Questions and answers

Subscribe to Our Youtube Channel

Related Manuals for Axon Axopatch 200B

Summary of Contents for Axon Axopatch 200B

  • Page 1 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...
  • Page 3 Changing the Fuse by the shipper, there is a good chance used under laboratory conditions. The Axopatch 200B uses a 250 V~, T2A, that the Axopatch 200B will shift within Operate in a clean, dry 5 x 20 mm fuse.
  • Page 4 1. Emploi à l'intérieur. boulettes de mousse pour protéger Changement du fusible 2. Fluctuations du réseaux l'Axopatch 200B. En cas de chute de la L'Axopatch 200B emploie un fusible de d'alimentation: ne doivent pas boîte durant son transport, l'Axopatch 250 V~, T2A, 5 × 20 mm.
  • Page 5 (“headstage”) wird über einen mit der Versand des Axopatch 200B VORGESEHEN, BEI MENSCHLICHEN Aufschrift “headstage” Bei dem Axopatch 200B handelt es sich VERSUCHEN VERWENDET ZU gekennzeichneten 15 Pin D- um ein solide gebautes Instrument, das WERDEN UND AUCH NICHT AN Unterstecker an der Rückwand des...
  • Page 6 En general, la mejor manera de empacar FABRICANTE SE PODRÍA PERDER LA Evite derramar líquidos sobre el el Axopatch 200B es en la caja original PROTECCIÓN PROVISTA POR EL “headstage”. de fábrica. Si ésta ya no se encuentra EQUIPO.
  • Page 7 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 de protection Schutzleiterpol Terminal de conductor protector Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 8 INFORMACION IMPORTANTE Axopatch 200B, Copyright March 1997-1999, Axon Instruments, Inc.
  • Page 9 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 HUMANS IN ANY WAY. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 10 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. Axopatch 200B, Copyright March 1997-1999, Axon Instruments, Inc.

  • Page 11: Table Of Contents

    Brief Method For Setting Series Resistance Compensation.........19 Detailed Method For Setting Series Resistance Compensation ......22 Current Clamp (Model Cell)..................28 Single-Channel Recording (Real Cell) .................30 Whole-Cell Recording (Real Cell) ................35 CHAPTER 4: INTERFACING A COMPUTER TO THE AXOPATCH 200B ....41 Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 12 Mounting the Headstage ..................55 Cleaning ....................... 55 Static Precautions....................56 Optical Pick-up ....................56 Acoustic Pick-up ....................56 Tuning the Headstage................... 56 Holders......................... 57 Features ........................ 57 Parts........................58 Use ........................59 Glass Dimensions....................61 Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 13 If the Probe Output is Slow, How Can Voltage Clamping Be Fast? ....78 What is Clamped During Voltage Clamping? ..........79 Capacitance Compensation...................79 Pipette Capacitance Compensation...............79 Whole-Cell Capacitance Compensation ............81 Absolute Value....................83 Limitations ....................83 Series Resistance ......................83 Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 14 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 Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 15: List Of Figures And Tables

    FIGURES AND TABLES • vii LIST OF FIGURES AND TABLES Page Figure 1 Connections for testing Axopatch 200B and PATCH-1U model cell....6 Figure 2 Pipette capacity compensation ................15 Figure 3 Whole-cell capacity compensation..............19 Figure 4 Maximum % PREDICTION as a function of voltage step........ 21 Figure 5 Series resistance compensation, brief method...........
  • Page 16 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 Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 17: Chapter 1: Introduction

    Much of the circuitry of the Axopatch 200B is devoted to eliminating the transients introduced by these resets. In most circumstances, the transient elimination is so good that the resets will not affect the recording and the benefits of the capacitor feedback will be available without penalty.
  • Page 18 We have designed the Axopatch 200B with great care and we are confident that it is an excellent tool. But the benefits of this instrument will not be realized if the user is not well versed in its operation and at the same time knowledgeable about the experimental techniques.

  • Page 19: Chapter 2: Functional Checkout

    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 power cable, b) the headstage, c) a cable from the SCALED OUTPUT BNC to one channel of the oscilloscope.
  • Page 20 4 • FUNCTIONAL CHECKOUT 2) Set the front panel controls of the Axopatch 200B as follows: PIPETTE OFFSET: About 5.0 0.5 ms PIPETTE CAPACITANCE COMP.: Minimum (fully counterclockwise) SERIES RESISTANCE COMP. % PREDICTION: 0 %, OFF SERIES RESISTANCE COMP. % CORRECTION: 0 %, OFF SERIES RESISTANCE COMP.
  • Page 21 FUNCTIONAL CHECKOUT • 5 4) Turn the meter switch to I . Note the reading on the rms noise display for the three headstage configurations (toggle the configuration between the PATCH and WHOLE CELL positions). The expected values under optimal conditions are: CV 203BU PATCH β...
  • Page 22 6 • FUNCTIONAL CHECKOUT Test WHOLE CELL and PATCH configurations: Figure 1. Connections for testing Axopatch 200B and PATCH-1U model cell. 6) Remove the foil shield temporarily. Connect the black lead (2 mm pin at one end) to the PATCH-1U model cell ground (2 mm socket at central position) then connect this lead to the ground input of the headstage (1 mm pin).
  • Page 23 FUNCTIONAL CHECKOUT • 7 This clamps 10 mV across the 10 MΩ resistor in the model cell BATH position. The panel meter should read the correct current (1 nA) within error limits (2% for the meter, 1% for the resistor and 1% for the HOLDING COMMAND generator plus errors for zeroing, etc.).
  • Page 24 +2.6 nA. 21) Set the ZAP control to 0.5 ms. Depress the ZAP button several times. The OVLD light in the SCALED OUTPUT section should flash each time the button is pushed. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 25 FUNCTIONAL CHECKOUT • 9 Test TEMPERATURE CONTROL: 22) Set HEADSTAGE COOLING switch to OFF (switch is located on back panel). Set Meter Switch to TEMP. 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).

  • Page 27: Chapter 3: Use Of The Patch Clamp - A Tutorial

    Use of the Patch Clamp — A Tutorial The purpose of this chapter is to ease you into the use of your Axopatch 200B. The controls have been carefully grouped for clarity. Many of them can be switched off and ignored until you become more familiar with the instrument and patch clamping.

  • Page 28: Single Channel Recording (Model Cell)

    12 • USE OF THE PATCH CLAMP – A TUTORIAL Single Channel Recording (Model Cell) Set the front panel controls of the Axopatch 200B as follows: PIPETTE OFFSET: About 5.0 PIPETTE CAPACITANCE COMP. Minimum (fully counterclockwise) ZAP: 0.5 ms SERIES RESISTANCE COMP. % PREDICTION: 0 %, OFF SERIES RESISTANCE COMP.

  • Page 29: Using The V-Clamp Mode

    USE OF THE PATCH CLAMP – A TUTORIAL • 13 in the model cell by the 5 mV SEAL TEST command. The total height of the pulse is about 5 volts. (Note: With a 10 MΩ resistance a 5 mV step will generate 0.5 V; the x10 gain provides a 10-fold amplification resulting in a 5 V output).

  • Page 30: Adjustment Of Pipette Capacitance Compensation

    LEAK SUBTRACTION control. Now change the HOLDING COMMAND switch from "+" to "-". If the LEAK SUBTRACTION circuit is properly adjusted, the current trace will stay on zero as the HOLDING COMMAND is switched back and forth between "+" and "-". Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 31: Whole-Cell Recording (Model Cell)

    SEAL TEST ON 5kHz FILTER 1 1 1 1 m m m m s s s s Figure 2. Electrode (pipette) capacitance compensation. Whole-Cell Recording (Model Cell) Set the front panel controls of the Axopatch 200B as follows: PIPETTE OFFSET: About 5.0 ZAP: 0.5 ms...

  • Page 32: Pipette Offset Adjustment

    2 V to the EXT. COMMAND input. (Alternatively, if a pulse generator is not conveniently available you can use the SEAL TEST.) Trigger the scope from the external source. This will produce large transients followed by steps in Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 33 USE OF THE PATCH CLAMP – A TUTORIAL • 17 the command potential of 40 mV amplitude and about 10 ms duration. If you are using the SEAL TEST it will generate a 5 mV command instead of a 40 mV command. You should scale the amplitude values in the following paragraphs accordingly.
  • Page 34 18 • USE OF THE PATCH CLAMP – A TUTORIAL *** Insert figure 3 near here *** Figure 3. Whole-cell capacitance compensation. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 35: Series Resistance Compensation

    USE OF THE PATCH CLAMP – A TUTORIAL • 19 Series Resistance Compensation The Axopatch 200B is capable of series-resistance compensation equal to that of the Axopatch 200A and substantially better than has been possible with any other commercial patch clamp to date. In order to achieve the outstanding performance of the Axopatch 200B it is critical to set its controls properly.
  • Page 36 SERIES RESISTANCE control may be required, followed by further readjustments of FAST MAG and FAST τ. An iterative procedure works best. It should be possible to reach this high degree of compensation without any residual transient as shown in Figure Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 37 USE OF THE PATCH CLAMP – A TUTORIAL • 21 *** insert fig. 5 near here *** Figure 5. Series resistance compensation, brief method. Chapter 3...

  • Page 38: Detailed Method For Setting Series Resistance Compensation

    COMPENSATION. The amplitude of the initial negative-going component of the residual transient will decrease. Continue to change the control setting until the waveform of the residual transient changes to a monophasic positive going response. At the point where the negative - pA peak-to-peak Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 39 USE OF THE PATCH CLAMP – A TUTORIAL • 23 component has just been eliminated, you may observe a small wiggle at the leading edge of the residual transient. Continue to reduce the FAST MAG control until the leading edge of the transient is smooth and "S-shaped".
  • Page 40 WHOLE CELL CAP. potentiometer setting to make the residual transient disappear into the noise (Figure 6h). If this is not sufficient, very small readjustments of the SERIES RESISTANCE control and FAST MAG and FAST τ may also be required. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 41 USE OF THE PATCH CLAMP – A TUTORIAL • 25 *** insert fig. 6 near here ** Figure 6. Series resistance compensation, detailed method. Chapter 3...
  • Page 42 26 • USE OF THE PATCH CLAMP – A TUTORIAL *** insert fig. 6 near here *** Figure 6. Series resistance compensation, detailed method (cont.) Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 43 USE OF THE PATCH CLAMP – A TUTORIAL • 27 At this point, 95% of the approximately 10 MΩ series resistance has been compensated; the residual series resistance is 500 kΩ. An ionic current of 2 nA amplitude would now cause only a 1 mV error in the membrane potential relative to the command potential, i.e., a 20-fold reduction from the situation prior to the use of CORRECTION.

  • Page 44: Current Clamp (Model Cell)

    Now is the time to set the external command signal and holding current command desired when you go into I- CLAMP mode. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 45 USE OF THE PATCH CLAMP – A TUTORIAL • 29 Set the external command for a 5 Hz, 100 mV rectangular waveform. This will cause a 200 pA current to be forced through the model cell. Switch the MODE to I-CLAMP NORMAL.

  • Page 46: Single-Channel Recording (Real Cell)

    OUTPUT will allow you to assess the fidelity of the clamp, and to determine whether I-CLAMP FAST or I-CLAMP NORMAL is more suited to a particular cell and electrode combination. Single-Channel Recording (Real Cell) Set the front panel controls of the Axopatch 200B as follows: PIPETTE OFFSET: About 5.0 ZAP: 0.5 ms...
  • Page 47 USE OF THE PATCH CLAMP – A TUTORIAL • 31 electrode and the patch electrode will show up as a non-zero tracking voltage on the meter. Adjust the PIPETTE OFFSET potentiometer until V is zero. (Some investigators TRACK prefer not to use the tracking circuit but rather do their early adjustments and seal formation in voltage clamp mode.
  • Page 48 Once a gigohm seal is established, the rectangular current pulse will disappear entirely and be replaced by capacitance transients in synchrony with the rising and falling edges of the command pulse (Figure 8, lowest trace). Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 49 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 the HOLDING COMMAND circuit included in the Axopatch 200B. Turn the...
  • Page 50 1 GΩ seal and a 100 mV HOLDING COMMAND, the seal current will be 100 pA. If, for example, you have set the overall Axopatch 200B gain so that you can look at a 5 pA channel current taking up one division at 1 V/div on the oscilloscope screen, the application of this HOLDING COMMAND will cause the current trace to go right off the screen.

  • Page 51: Whole-Cell Recording (Real Cell)

    USE OF THE PATCH CLAMP – A TUTORIAL • 35 Whole-Cell Recording (Real Cell) Set the front panel controls of the Axopatch 200B as follows: PIPETTE OFFSET: About 5.0 ZAP: 0.5 ms SERIES RESISTANCE COMP. % PREDICTION: 0 %, OFF SERIES RESISTANCE COMP.
  • Page 52 36 • USE OF THE PATCH CLAMP – A TUTORIAL The Axopatch 200B contains a ZAP circuit to aid in breaking into the cell. This circuit delivers a pulse of 1.3 VDC volts for variable durations ranging from 0.5 to 50 ms. The user sets the duration which is optimal for the particular cells in use by adjusting the potentiometer that makes up the outer part of the control.
  • Page 53 USE OF THE PATCH CLAMP – A TUTORIAL • 37 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.
  • Page 54 This procedure leads to unique settings of the SERIES RESISTANCE and WHOLE CELL CAP. controls corresponding to the electrode and cell being clamped. When the transient is minimized, the access resistance (pipette resistance and any resistive contribution from Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 55 IR drop across the access resistance. The proper adjustment of these controls is discussed 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.

  • Page 57: Chapter 4: Interfacing A Computer To The Axopatch 200B

    Interfacing a Computer to the Axopatch 200B The Axopatch 200B has many features that allow extensive interactions with a laboratory computer. Some are inputs and some are outputs. This section describes the use of these features with a computerized patch clamp setup.
  • Page 58 The user is free to implement some scheme to interrogate this line during data collection to determine precisely when the data is not valid. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 59: Chapter 5: Low Noise Recording Techniques

    Chapter 5 Low Noise Recording Techniques The PATCH configuration of the Axopatch 200B is capable of producing single-channel recordings with significantly lower noise than a standard resistive patch clamp because of the inherently low noise of the integrating headstage, particularly at low to moderate frequencies (below 10 kHz).

  • Page 60: Glass Type And Coating

    The deviation from 90° is the "loss factor". The loss factor is related to the power dissipated in the dielectric. Since energy is lost in the dielectric, dielectrics (e.g., glasses) are commonly referred to as "lossy". Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 61 LOW NOISE RECORDING • 45 Even if one uses electrically superior glasses, low noise will not be obtained unless the outer surface of the glass is coated with a hydrophobic substance, such as Dow Corning Sylgard #184. This substance prevents the bathing solution from creeping up the outer wall of the pipette glass.
  • Page 62 Kovar seal borosilicate 3320 1.50 Tungsten seal borosilicate 7050 1.60 Series seal borosilicate KG-33 2.20 Kimax borosilicate 7740 2.60 Pyrex borosilicate 1720 2.70 11.4 Aluminosilicate N-51A 3.70 Borosilicate 5.10 Soda lime 0080 6.50 Soda lime Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 63: Seal

    LOW NOISE RECORDING • 47 The holders supplied with the Axopatch 200B are made of polycarbonate. Polycarbonate was experimentally found to produce the lowest noise among ten substances tested. It was only slightly better than polyethylene, polypropylene, and Teflon, but was much better than nylon, Plexiglass, and Delrin.

  • Page 64: Signal Generator

    One last potential noise 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.

  • Page 65: Chapter 6: Reference Section: Instrument Operation

    The major topics in this section are organized alphabetically. Bridge Mode The Axopatch 200B may be used to follow cell membrane potential, in a way similar to the bridge mode of conventional microelectrode amplifiers, when in current clamp (I=0, I-CLAMP NORMAL and I-CLAMP FAST; please see Current Clamp section below).

  • Page 66: Command Potentials

    β = 1 β = 0.1 β = 1 ± 200 mV ± 200 mV ± 1 V ± 1 V CLAMP ± 20 nA ± 2 nA ± 100 nA ± 10 nA CLAMP Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 67: Seal Test

    50 pA (β = 1) or 500 pA (β = 0.1). Current Clamp The Axopatch 200B can be used in current-clamp mode similarly to a conventional microelectrode amplifier. Series Resistance compensation is functional in this mode.

  • Page 68: Whole-Cell Parameters

    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 listed in the chapter for current clamp.

  • Page 69: Limitations

    Series Resistance Compensation. However, similar to many other patch clamps, current clamp in the Axopatch 200B is not as fast or as stable as current clamp in a conventional microelectrode amplifier such as the Axoclamp or the Axoprobe. This difference in current-clamp performance results from significant differences in the design of the headstages.

  • Page 70: Electrochemistry

    54 • INSTRUMENT OPERATION Electrochemistry The Axopatch 200B may be used to perform electrochemical measurements. Typically, voltammetric experiments require a large command potential range in voltage-clamp mode. This may be accomplished by setting the Holding Command toggle to "x5" for internal commands and by using the 100 mV/V Rear-Switched external command input for external commands.

  • Page 71: Frequency Boosting

    INSTRUMENT OPERATION • 55 Frequency Boosting The WHOLE CELL mode of the Axopatch 200B uses a resistor in the headstage feedback that requires frequency boosting (tuning) for optimal performance. The frequency boosting circuit is tuned at the factory and will usually not require readjustment in the field. If tuning is required, see the (page 121).

  • Page 72: Static Precautions

    (i.e., you hear and feel crackles when you touch things), you should touch a grounded metal object immediately before touching the headstage. You should not switch off power to the Axopatch 200B when handling the headstage input since this will upset thermal equilibrium.

  • Page 73: Holders

    90° from the headstage. The HL-U holder is designed to be used with Axon Instruments amplifiers, and fit all U-type CV and HS series of headstages. These headstages have a threaded white Teflon collet.

  • Page 74: Parts

    2 mm lengths, the silicone tubing will yield approximately 30 replacement silicone seals. Additional pipette caps, cone washers, silicone tubing, pins and silver wire can be purchased from Axon Instruments, as well as optional Ag/AgCl pellet assemblies. Optional Ag/AgCl Pellets The HL-U holder will accommodate a 1 mm diameter Ag/AgCl pellet that should provide many months of DC-stable recordings.

  • Page 75: Use

    INSTRUMENT OPERATION • 59 Figure 12. Ag/AgCl pellet assembly. Insertion of Pipette Make sure the electrode cap is loosened so that pressure on the cone washer is relieved, but do not remove the pipette cap. Push the back end of the pipette through the pipette cap and cone washer until it presses against the pipette seat.
  • Page 76 For easy-to-use recipes see Microelectrode Methods for Intracellular Recording and Ionophoresis, by R.D. Purves, London: Academic Press, 1981, p. 51. The Axon Guide. Foster City, CA: Axon Instruments, Inc., 1993, p. 83. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 77: Glass Dimensions

    HLR-U right-angle adapters allow the HL-U series holder to emerge at 90° from the headstage. Use the HLR-U with the HL-U holder. HLB-U BNC-to-Axon adapter allows conventional BNC-type holders to be used with Axon 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).
  • Page 78 EXTERNAL COMMAND INPUT (front panel switched) Applies external command signal divided by 50 to the command input of the Axopatch 200B when the front toggle switch is set to EXT. COMMAND. EXTERNAL COMMAND INPUT (rear panel switched) Applies external command signal divided by 10 to the command input when the rear panel switch is engaged.

  • Page 79: Output Section

    INSTRUMENT OPERATION • 63 Output Section All the controls in the Output Section affect only the signal on the Scaled Output. Filter The filter is a 4-pole lowpass Bessel filter. The attenuation of signals and noise above the - 3 dB frequency is 80 dB/decade (24 dB/octave). The Bessel characteristic is suitable for patch and voltage clamping because it introduces <...

  • Page 80: Output Bncs

    CELL configuration. To achieve the best compromise between noise and dynamic 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.
  • Page 81 INSTRUMENT OPERATION • 65 FREQUENCY TELEGRAPH Provides a series of voltages that can be read by a computer to determine the setting on the filter switch. The voltages and their associated frequency settings are as follows: Frequency (kHz): Frequency Telegraph Voltage Output (V): 2 GAIN TELEGRAPH Provides a series of voltages that can be read by a computer to determine the α...
  • Page 82 = pipette current in nA CORR = % correction set on SERIES RESISTANCE CORRECTION control = resistance in MΩ set on SERIES RESISTANCE control In TRACK mode, 10 V is ten times the actual potential. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 83: Panel Meter

    INSTRUMENT OPERATION • 67 In I-CLAMP mode, if the current is zero, 10 V is ten times the actual membrane potential. If current is flowing in the pipette, 10 V includes the voltage drop across the pipette. The actual membrane potential is given by the following equation: 10 V _______...

  • Page 84: Power-Supply Voltage Selection And Fuse Changing

    . No range switching is required. Changing the Fuse The Axopatch 200B uses a 2.0 A, 250 V T 2A 5 x 20 mm fuse. In the event of fuse failure, disconnect the power cord. Before changing the fuse investigate the reason for its failure.

  • Page 85: Zap

    INSTRUMENT OPERATION • 69 In order to go from cell-attached patch clamping to whole-cell patch clamping it is necessary to rupture the patch. This is normally done by carefully controlled suction. Another, easier to apply, technique for rupturing the patch is ZAP. ZAP works by applying a large hyperpolarizing voltage (1.3 V ) to the patch for a controlled duration.

  • Page 87: Chapter 7: Reference Section: General Information

    1) Ground the preparation 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.

  • Page 88: Model Cell

    4) Try grounding auxiliary equipment from a ground distribution bus. This bus should be connected to the Axopatch 200B via the yellow banana (4 mm) socket on the rear panel. This socket is connected to the signal ground of the Axopatch 200B (i.e., the outer conductors of all the BNC connectors).

  • Page 89: Model Bilayer

    GENERAL INFORMATION • 73 Ω Ω PATCH PATCH 3.3 pF + STRAY 5 pF STRAY Ω 500M CELL Ω CELL 500M Ω Ω 33 pF 33 pF Ω BATH BATH 4 pF ACTUAL CIRCUIT EQUIVALENT CIRCUIT Figure 13. PATCH-1U model cell. Model Bilayer The MCB-1U model bilayer contains a 10 kΩ...

  • Page 90: Power-Supply Glitches

    74 • GENERAL INFORMATION Power-Supply Glitches The Axopatch 200B has been designed to minimize the effects of power-supply transients (glitches). Some power-supply glitches do, however, get through. These can cause transients to appear on the voltage and current outputs which may corrupt high-sensitivity recordings.

  • Page 91: Chapter 8: Reference Section: Principles Of Operation

    PRINCIPLES OF OPERATION • 75 Chapter 8 Reference Section: Principles of Operation Headstages Principals of Operation Patch-clamp headstages are current-to-voltage (I-V) converters. That is, 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.

  • Page 92: Capacitor Feedback

    Figure 16 shows the essential parts of an integrating headstage. INTEGRATOR DIFFERENTIATOR = 1 pF OFFSET SCALING Figure 16. Capacitive headstage. 1 op amp - operational amplifier Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 93 With the U430 transistors used in the Axopatch 200B, this low frequency noise can be substantially less than that of the 50 GΩ feedback resistor customarily used in resistive headstages.

  • Page 94: If The Probe Output Is Slow, How Can Voltage Clamping Be Fast

    NOT VALID Figure 17. Signal handling during resets. The Axopatch 200B contains both a resistive and capacitive feedback 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.

  • Page 95: What Is Clamped During Voltage Clamping

    PRINCIPLES OF OPERATION • 79 In fact, despite the slow current output of the I-V converter, the voltage clamp of the pipette is rapid. The pipette is connected to the negative input (summing junction) of the op amp. The command potential is connected to the positive input of the op amp.
  • Page 96 . A small amount of can only be represented as a capacitor with a series resistance component. This takes longer to charge to its final value and is compensated by the SLOW controls. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 97: Whole-Cell Capacitance Compensation

    PRINCIPLES OF OPERATION • 81 Whole-Cell Capacitance Compensation The SERIES RESISTANCE and WHOLE-CELL CAP. controls are used to charge the membrane capacitance (C ). Figure 19 is a simplified circuit of these controls. Figure 19. Whole-cell capacitance compensation circuit. Assume that the fast and slow electrode compensation controls have already been set to compensate for C .
  • Page 98 The I and V outputs on the Axopatch 200B show the I and V trace illustrated in Figure 20. It is easy to mistakenly think that the time course for charging the membrane is very fast but this is clearly not the case.

  • Page 99: Absolute Value

    1 cm membrane is 1 µF. The setting of this control is available for computer acquisition on the CELL CAPACITANCE output telegraph on the rear panel of the Axopatch 200B. Limitations The measurement of series resistance (R ) and cell capacitance (C...
  • Page 100 τ , where C is the cell membrane capacitance. This time constant is 330 µs for the model cell provided with the Axopatch 200B (R = 10 MΩ, C = 33 pF). This means that the actual membrane potential response to a step voltage command will have a 10-90% risetime of more than 0.7 ms and will not settle to within 1% of its final...
  • Page 101 PRINCIPLES OF OPERATION • 85 The Axopatch 200B uses a dual approach for the correction of the above errors associated with series resistance. In this regard, the performance of the Axopatch 200B is unparalleled by any other commercial patch clamp.
  • Page 102 (1 - %PREDICTION/100), and, in the frequency domain s = jw (w is the natural frequency, w = 2πf), or in the time domain s is the operator τ d/dt. Thus, V (1 + (R -1)e Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 103: Saturation Effects

    PREDICTION circuit itself). The command plus PREDICTION signal is attenuated at the headstage by a 10:1 voltage divider. Since the circuitry in the Axopatch 200B mainframe will saturate at about ±11-12 V, V is limited in absolute value to about 1.1 to 1.2 V.
  • Page 104 , where τ -3 dB bandwidth of this filter is given by 1/2πτ is the setting (in seconds) of the LAG control. For example, a LAG of 5 µs corresponds to filtering the CORRECTION Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 105 PRINCIPLES OF OPERATION • 89 signal at 32 kHz; 10 µs corresponds to 16 kHz, 20 µs corresponds to 8 kHz, etc. The LAG control is used to ensure stability when large amounts of CORRECTION are used. It is generally good practice to begin using CORRECTION with the LAG control set at 10-20 µs or more.

  • Page 106: Oscillations

    90 • PRINCIPLES OF OPERATION for ionic currents; this is not true in the Axopatch 200B. On the other hand, in some cases it might be impossible to advance the CORRECTION percentage beyond about 70% without causing instability. Nevertheless, PREDICTION, which is inherently stable up to...

  • Page 107: Pipette Offset

    PRINCIPLES OF OPERATION • 91 For best results, the cell membrane resistance should be many fold higher than the pipette resistance. This is normally the case for cells at rest carrying small drug- activated or synaptic currents. However, during voltage activation the cell membrane resistance could fall a hundredfold or more to values similar to or less than the series resistance.

  • Page 108: Leak Subtraction

    I trace from jumping into saturation. I is severely distorted during TRACK mode; the effect is similar to AC coupling. Note: The Axopatch 200B should never be left in TRACK mode once data is being recorded.

  • Page 109: Zap

    Also, because the Axopatch 200B is stable while driving purely capacitive loads (up to 1000 pF), one has the ability of minimizing noise by minimizing access resistance.
  • Page 110 94 • PRINCIPLES OF OPERATION When the Axopatch 200B probe is in the PATCH configuration, it has a 1 pF capacitor as its feedback element (Figure 21). 10 kΩ 1 pF INTEGRATOR STEP Figure 21. Integrator driving bilayer model. When a voltage step, V...

  • Page 111: Noise Vs. Access Resistance

    Noise vs. Access Resistance The Axopatch 200B is quite comfortable with loads of up to 1000 pF of pure capacitance (the maximum bandwidth decreases to about 20 kHz, no overshoot). This can be used to great advantage when doing bilayer experiments; the lower the access resistance, the lower the noise.

  • Page 112: Current And Voltage Conventions

    SERIES RESISTANCE (ohms) Figure 22. Typical current noise in bilayer experiments. Current and Voltage Conventions The terminology used in 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.
  • Page 113 PRINCIPLES OF OPERATION • 97 Positive Potential The term positive potential means a positive voltage at the headstage input with respect to ground. Transmembrane Potential The transmembrane potential (V ) is the potential at the inside of the cell minus the potential at the outside.
  • Page 114 This approach has been rejected by Axon Instruments because of the real danger that if the researcher forgets to move the switch to the preferred position, the data recorded on the computer could be wrongly interpreted.
  • Page 115 PRINCIPLES OF OPERATION • 99 Patch Clamp By design, the patch-clamp command voltage is positive if it increases the potential inside the micropipette. Whether it is hyperpolarizing or depolarizing depends upon whether the patch is "cell attached", "inside out" or "outside out". The patch-clamp pipette current is positive if it flows from the headstage through the tip of the micropipette into the patch membrane.
  • Page 116 Whole-cell voltage clamp outward membrane current Whole-cell current clamp outward membrane current 2) A positive shift in the command potential is: Cell-attached patch hyperpolarizing Inside-out patch hyperpolarizing Outside-out patch depolarizing Whole-cell voltage clamp depolarizing Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 117: Troubleshooting

    Whole-cell voltage clamp Troubleshooting It has been our experience at Axon Instruments that the majority of troubles reported to us have been caused by faulty equipment connected to our instruments. If you have a problem, please disconnect all instruments connected to the Axopatch 200B except for the headstage.

  • Page 119: Chapter 9: Specifications

    SPECIFICATIONS • 103 Chapter 9 Specifications Unless otherwise noted: T = 20 °C, 1 hr warm-up time. CV 203BU Headstage Construction 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...
  • Page 120 Test signal applied via SPEED TEST input; PATCH mode: Internal: 140 kHz Max. External: 100 kHz (limited by output filter) Capacitive Load Stability 1000 pF, 0 Ω in series FET - Field Effect Transistor Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 121 SPECIFICATIONS • 105 Maximum Instrument Noise Measured with minimal external noise sources (i.e., radiated line frequency noise, mechanical vibration), 8-pole Bessel filter. PATCH WHOLE CELL β = 1 β = 0.1 Line freq. Without holder: & harmonics 0.005 pAp-p 0.005 pAp-p 0.005 pAp-p 0.1-100 Hz 0.030 pAp-p...
  • Page 122 Figure 23B. Typical broadband current noise spectrum, patch mode. TOTAL NOISE (pA RMS) 0.01 0.001 100 Hz 10 kHz 1 kHz BANDWIDTH Figure 23C. Typical total current noise as a function of bandwidth, patch mode. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 123 SPECIFICATIONS • 107 Reset Characteristics (Patch Mode only) Total reset time 50 µs ± 10%. This includes: integrator reset 10 µs differentiator reset 30 µs other overhead 10 µs Time between resets (T For DC currents: T = 10/(I BIAS where I and I are in pA and T...
  • Page 124 0 - 10 pF Slow τ: 0.1 - 10 ms Slow Magnitude: 0 - 1 pF These controls are used to charge pipette capacitance. In I-CLAMP modes they act as a negative capacitance. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 125 SPECIFICATIONS • 109 Whole-Cell Capacitance β = 1: 0.3 - 100 pF β = 0.1: 3 - 1000 pF Series Resistance 0 - 100 MΩ These controls are used to charge membrane capacitance in whole-cell voltage clamp. For PATCH mode, whole-cell capacitance is not operative. In I-CLAMP modes only the SERIES RESISTANCE control is operative.
  • Page 126 FAST mode stable for electrode resistances greater than 10 MΩ. SERIES RESISTANCE control is active. Slow I-CLAMP to zero current. Track Slow I-CLAMP to zero current used to correct pipette offset. Selected mode sets analog voltage on MODE TELEGRAPH OUTPUT. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 127 SPECIFICATIONS • 111 Amplitude +1.3 V at pipette for chosen duration. Duration 0.5-50 ms or Manual. Triggered by front-panel pushbutton. In Manual position ZAP amplitude is maintained as long as pushbutton is depressed. Command Potentials Seal Test 5 mV command at line frequency (voltage clamp mode) 50 pA (current clamp mode, β...
  • Page 128 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 Reset Positive edge triggered. Initiates a reset of the integrator; has no control over the duration of reset. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 129 SPECIFICATIONS • 113 Blank Activate Causes SCALED OUTPUT and I OUTPUT to hold their initial value for the duration of the blanking pulse. Does not affect 10V output. Speed Test Injects current into headstage input through a 1 pF capacitor. Injected current waveform is the derivative of the voltage waveform applied at SPEED TEST input.
  • Page 130 10 100 Telegraph Output (V): Mode I-CLAMP I-CLAMP Mode: TRACK V-CLAMP I = 0 NORMAL FAST Scaled Output: Telegraph (volts): 4 Applicable for β = 0.1 only Applicable for β = 1 only Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 131 SPECIFICATIONS • 115 Cell Capacitance 0 to +10 V, proportional to setting 0-100 pF (for β = 1; 0-1000 pF for Telegraph Output: β = 0.1 ) when WHOLE CELL CAP. switch is in the ON position. 0 to -10 V, when WHOLE CELL CAP. switch is in the OFF position. Data Not Valid Output goes High during a reset in PATCH mode or for the duration of a BLANK ACTIVATE pulse in either PATCH or WHOLE CELL mode.
  • Page 132 100 to 240 V Universal voltage input. Line Frequency: 50-60 Hz. Power 30 W Fuse 0.5 A slow. 5 x 20 mm. Line Filter: RFI filter is included. Line cord: Shielded line cord is provided. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 133 SPECIFICATIONS • 117 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 Chapter 9...

  • Page 135: Chapter 10: References

    REFERENCES • 119 Chapter 10 References Fidler, N., and Fernandez, J.M. Phase tracking: an improved phase detection technique for cell membrane capacitance measurements. Biophysical Journal 56:1153-1162, 1989. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv, 391, 85-100, 1981.

  • Page 137: Chapter 11: Headstage Tuning Procedure

    TUNING THE HEADSTAGE • 121 Chapter 11 Headstage Tuning Procedure This procedure should be carried out if the user has done any of the following: Purchased an additional headstage and needs to match it to the main unit. Has found the step response of the WHOLE CELL configuration is unacceptable. This should be verified by applying a high quality triangle voltage waveform to the SPEED TEST BNC.
  • Page 138 CELL CONFIG. Set CONFIG. to WHOLE CELL β = 1. Set HOLDING COMMAND to 200 mV. Switch HOLDING COMMAND between + and - . Trim RT14 until meter reads zero for both polarities. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 139 TUNING THE HEADSTAGE • 123 Frequency Tuning (Whole Cell Config.) Set CONFIG. to WHOLE CELL β = 1. Set OUTPUT GAIN to x1. Set FILTER to 100 kHz. Connect scope to SCALED OUTPUT BNC. Trigger scope from waveform generator. Set scope to 0.2 V/div, 1 ms/div. Apply a 10 V 100 Hz triangle wave to the SPEED TEST BNC.
  • Page 140 FILTER to 5 kHz. Scope to 0.2 V/div, 0.5 ms/div. Trigger scope from DATA NOT VALID BNC. Connect pulse generator to FORCED RESET BNC. Set pulse generator +5 V ( > 100 µs duration), 100 Hz. Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.
  • Page 141 Turn switch 1 of SW8 to ON. Trim RT22 (TAIL TAU) and RT23 (TAIL MAG) to minimize transient Internal Reset Remove pulse generator from Axopatch 200B. Connect PATCH-1U model cell in PATCH position to headstage. Set FILTER to 10 kHz.
  • Page 142 Trim RT25 and RT26 to minimize slowest transient. As above, some re-trimming of RT20 and RT21 may be necessary to achieve optimum compensation. See Figure 25e. Figure 25. Reset transient compensation. For clarity, only one reset polarity is shown (positive DC current). Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 143: Index

    INDEX • 127 INDEX Access Resistance, 83, 95 External Command, 62, 72 Acoustic Pick-up, 56 Filter Bilayers, 93 See Output, 63 Noise, 94 Forced Reset, 62 Blank Activate, 62 Frequency Telegraph, 64 Bridge Mode, 49 Fuse Changing, 68 Capacitance Compensation Gain Current Clamp, 51 See Output, 63...
  • Page 144 Trouble Shooting, 101 Panel Meter, 67 Percentage Compensation Voltage Convention, 96 See Series Resistance, 83 Perforated Patch, 36 Whole-Cell Recording Pipette, 55 Model Cell, 15 Chloriding, 60 Real Cell, 35 Cleaning, 59 Zap, 69, 93 Axopatch 200B, Copyright 1997-1999, Axon Instruments, Inc.

  • Page 145: Warranty

    WARRANTY • 129 WARRANTY We warrant every Axopatch 200B and every headstage to be free from defects in material and workmanship under normal use and service. For 12 months from the date of receipt, we will repair or replace without cost to the customer, any of these products that are defective and that are returned to our factory properly packaged with transportation charges prepaid.

  • Page 147: Warning

    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, Axon Instruments can ship the proper packaging material to you for a small fee.

  • Page 149: Circuit Diagrams Request

    Declaration Please send me the circuit diagrams and parts lists for the Axopatch 200B. I agree that I will only use the circuit diagrams and parts lists for service of the Axopatch 200B. I will not use them to create equivalent or competing products.
  • Page 151 CIRCUIT DIAGRAMS REQUEST FORM • 135 Please fold out so that you may refer to this page while reading the manual. Circuit Diagrams Request Form...
  • Page 153 Circuit Diagrams Request Form...

Table of Contents

Save PDF