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    Articles

    Axopatch 200B

    Axopatch 200B

    The Axopatch 200B is the latest version of Axon Instruments’ premier patch-clamp amplifier incorporating the innovative Capacitor-Feedback technology that provides the lowest-noise single-channel recording available. We have improved the integrating patch clamp in the 200B with our proprietary technology, which includes cooling of the active elements in theamplifier to achieve the lowest possible electrical noise. No previous amplifier has offered such low noise. Positioning your electrode will also be easier with our new, slim headstage design that improves access to the preparation.

    Other enhancements include three recording configurations in a single headstage (one patch and two whole cell ranges, with capacitance compensation ranges of 100 pF and 1000 pF), increased voltage and current command ranges for electrochemical measurements, built-in capacitance dithering capability for capacitance measurements, and addition of series resistance compensation to the current clamp circuitry to improve performance. Seal Test now provides current steps in current clamp mode as well as voltage steps in voltage clamp mode. Leak Subtraction is now more sensitive in the most important resistance range. The new, slim headstage design improves electrode access to the preparation, and the recording bandwidth has been doubled to up to 100 kHz.

    The amazingly low open-circuit noise of 0.13 pA rms (10 kHz) increases to only 0.145 pA rms when a patchpipette holder is attached to the headstage input and the pipette capacitance is fully compensated (to eliminate capacitance charging transients). The power of capacitor-feedback technology is shown in Figure 1: capacitor feedback is clearly superior to resistive feedback; cooled capacitor-feedback technology is even better!

    Axopatch 200B-2

    Like its predecessors, the Axopatch 200B incorporates capacitive and resistive feedback elements in its headstage, providing the best possible performance for single-channel and whole-cell patch clamping. Convenient features include: ZAP (to rupture patches when going whole cell), dual-speed current clamp (to allow faster current clamping in small cells), HoldingCommand to set voltage commands in voltage clamp and current commands in current clamp, and a choice of three gain settings on the dedicated current output (for patch, whole-cell and loose-patch modes).

    Unparalleled Performance, Utility and Ease of Use Included: Efficient controls for whole-cell capacitance compensation, a unique "super-charging" form of seriesresistance compensation that complements the conventional "correction" form, and a variable LAG control; output gain with 1000-fold dynamic range; 4-pole Bessel filter; built-in leak subtraction; dual external- command inputs; versatile panel meter displaying Holding Command, rms current noise, membrane potential, tracking potential, current at the headstage input, and cooled circuitry temperature; telegraphic output of values for output gain, filter  frequency, headstage mode (gain) and measured cell capacitance; ZAP; and dual-speed current clamp. Also, with the Axopatch 200B the bath is grounded for convenience of use and straightforward addition of command an a compensation potentials.

    Integrating Headstage Mode - Quiet Single-Channel Recording With its unprecedented low noise, the Axopatch 200B capacitor-feedback integrating headstage is ideal for measuring sub-picoamp current signals. The capacitor- feedback circuitry measures current as the rate of voltage increase across the capacitor; the voltage across the capacitor is the integral of the current. In contrast, resistor-feedback circuitry measures current as a voltage drop across a feedback resistor. This has the drawback of requiring extremely high-value resistors that become less ideal as they become larger in value, with accompanying increases in their inherent noise.

    Axopatch 200B-3

    Noiseless capacitors, on the other hand, are easily manufactured. Thus, capacitor-feedback technology offers the lowest possible noise for single-channel recording.

    Through the use of this technology, the electronic circuit noise has been reduced to such a level that the noise inherent in the components themselves has become significant. The Axopatch 200B actively cools the critical circuit components to about -15 °C in order to reduce the thermal noise and so lowers the noisecontribution of the amplifier itself to the lowest levels possible. The Axopatch 200B is the ultimate low-noise amplifier.In the design of the integrating patch clamp we addressed two additional technological challenges that are encountered in the pursuit of ultra-low noise. First and foremost, the voltage across the capacitor cannot ramp forever in one direction. Eventually, the supply limits are reached and the electronics must reset to the starting point to continue ramping. During this reset, thesystem freezes the output at the last measured value. For  the best performance, this reset must be kept brief and must occur as infrequently as possible. The reset time of the Axopatch 200B is so short (50 ms) that users may never see a reset under most normal recording conditions. Second, to minimize the frequency of resets, the voltage range across the capacitor must be as large as possible. By using unique compensation circuitry, the Axopatch 200B manages to use the entire ±10 V range  of the power supply to charge the capacitor. This reduces the frequency of resets without incurring the penalty of longer reset times.

    BilayersHeadstages useful in artificial bilayer experiments must  be stable with large input-capacitance loads. The Axopatch 200B is rock solid with an input capacitance of 1000 pF. Furthermore, the integrating technology in the Axopatch 200B enables the bilayer voltage to be stepped very quickly. There are two reasons for this. First, the integrator has an inherently large dynamic range that allows it to transiently pass currents of several microamperes. Second, during large command voltage steps the integrator enters the reset state. In this state, the headstage continues to operate as a voltage-clamp amplifier but with a very low feedback resistance. For the few milliseconds that the integrator is in the reset state it can pass up to 1 mA to rapidly charge the bilayer capacitance.

    Resistive Headstage Mode - Superb Whole-Cell PerformanceIn whole-cell recording more current noise is produced by the cell and the environment than by the patch clamp amplifier. Hence, the benefits of a low-noise capacitor- feedback headstage cannot be effectively utilized in whole-cell mode. For this reason, the Axopatch 200B uses traditional resistor feedback headstage electronics for the whole-cell mode of patch clamp recording. The CV 203BU headstage includes two feedback resistors to provide a wide range of current-passing capacity in the whole-cell mode. The 500 MW feedback resistor (b = 1) provides both low noise and a large current passing ability (20 nA). For larger currents, one can switch to the 50 MW feedback-resistor (b = 0.1) to pass up to 200 nA.

    Series Resistance PredictionThere are two goals of series resistance compensation. The first is to step the membrane potential to an assigned value as rapidly as possible. One way to speed up this process is to transiently increase the size of the command voltage step (applying a method known as “supercharging”). This causes the charging curve to briefly assume the steeper rate appropriate for a larger step. This transient, high-charging rate is terminated when the membrane potential approaches the intended value. On the Axopatch 200B, supercharging is available through the Series Resistance PREDICTION control. The supercharging transient in the Axopatch 200B is automatically and conveniently determined by the user when the conventional wholecell capacitance controls are set. Prediction refers to our unique algorithm for calculating the added supercharging waveform. Note that this technique does not require the command to be a voltage step; other commands, such as a sine wave, will also be supercharged.

    Series Resistance CorrectionThe second goal of series resistance compensation is to measure the ionic currents. To eliminate error due to the voltage drop across the electrode and to improve the bandwidth of the recording, conventional Series Resistance CORRECTION is required. This technique employs positive feedback to increase the command potential by adding a signal proportional to the measured current to the command input of the headstage. Series Resistance Prediction and Correction may be used together or separately on the Axopatch 200B. The  controls are concentric and can be manipulated at the same time. Turning the controls together is quite similar to the series resistance percentage compensation found on other patch clamps. In experiments with real cells we have found that both the Series Resistance Prediction and Correction on the Axopatch 200B can be routinelyset to levels exceeding 90%, enabling fast and accurate whole-cell voltage clamping. A variable LAG control permits an even greater degree of series resistance compensation to be achieved, although not at the highest frequencies.

    Dual-Speed Current ClampThe Axopatch 200B employs a dual-speed current clamp to optimize speed and stability. The choice of speeds depends on pipette resistance. For pipette resistances above 10 MW, the I-Clamp Fast mode can be employed to obtain a rise time of 10 ms under many experimental conditions. The I-Clamp Normal mode guarantees stability for any pipette resistance above 1 MW.In addition, current clamp to zero current (I=0 mode) is available, as is a slower clamp to zero current (Track mode) useful for following slow changes in pipette offset while approaching a cell before seal formation. The Holding Command can be used to set a holding current in I-Clamp. Two external command inputs are provided to permit multiple command sources (for cell capacitance experiments, current clamp, etc.). In addition, the Axopatch 200B includes series resistance compensation in current clamp that allows for correction of voltage errors due to the pipette resistance.

    Pipette OffsetResearchers are putting ever more unusual solutions into patch pipettes, producing large offset potentials. The Axopatch 200B provides ±250 mV of offset potential to handle even the most adverse situations.

    Dual Command PotentialsTwo separate command potential inputs allow you tosum command input signals from two different sources. For instance, you may wish to constantly supply a low level sine wave command for evaluation of membrane capacitance. Large voltage steps may be mixed with this signal to construct a Cm vs. Vm relationship. One of these command potentials may be switched on from the front panel to access the external source. The other is switched separately on the back panel. The back panel command is scaled to afford greater range (up to ±1 V), and so is quite useful for electrochemical measurements.

    Seal TestA convenient seal test is built into the External Command potential front panel switch. The oscillator frequency is set to the line frequency for automatic synchronous triggering on any oscilloscope. Seal Test may be used in voltage clamp mode (5 mV pulse) or in current clamp mode (50 pA (b = 1) or 500 pA (b = 0.1)) pulses.

    Holding CommandThe Holding Potential potentiometer can be used to manually establish the cell holding potential for voltage clamp or a holding current for current clamp. The Holding Command of the Axopatch 200B has been enhanced over that of its predecessors with the addition of a x1 and x5 switch that allows you to choose either 0-200 mV or 0-1 V ranges. (In current clamp, the command response at b = 1 is 0-2 nA or 0-10 nA, andat b = 0.1 it is 0-20 nA or 0-100 nA.) An ON/OFFswitch can disable this control when an external command from a computer is used to establish the holding potential.

    Pipette Capacitance CompensationPipette Capacitance Compensation is in operation inboth voltage- or current-clamp modes. There are controls to establish the Magnitude and t of two time constants, Fast and Slow, allowing complete compensation of the pipette capacitance waveform. Magnitude and t are controlled by concentric knobs allowing easy and convenient adjustment of parameters for each time constant.

    Cell Capacitance CompensationThe Whole-Cell Capacitance control can compensate up to 100 pF (b = 1) or 1000 pF (b = 0.1) to allow recording from a large range of cell sizes. The control may be disabled by an ON/OFF switch to allow evaluation ofthe cell capacitance charging waveform at any time during an experiment.

    Capacitance DitheringUseful for cell membrane capacitance measurements, Capacitance Dithering may be enabled during a TTL-High level signal to Whole Cell Capacitance Dither input to effectively increase the observed cell capacitance by 100 fF (b = 1) or 1 pF (b = 0.1). It may be used in conjunction with the DR-1 Resistance Dither unit (supplied with the Axopatch 200B) which normally provides a short-circuit link between preparation and ground. The DR-1 inserts a 500 kW resistor in series with bath ground during a TTL- High signal and is suitable for finding the phase tracking angle in capacitance measurement experiments.

    Output GainTen gain settings spanning a 1000-fold range may be selected to scale the output to the most desirable level. The Axopatch 200B output swing is ±10 V, providing the greatest compatibility to the vast range of recording devices. However, the dynamic range of the instrumentat unity gain is greater than the output swing. Thus a gain value of 0.5 is available to take advantage of the full dynamic range of the Axopatch 200B headstage. (Note: The full range is also faithfully reported by thefront panel meter.)

    Low-Pass FiltersThe scaled output passes through a 4-pole Bessel filter. The five settings from 1 kHz up to 100 kHz allow you toset the band pass appropriate to your application over a range double that previously available.Panel MeterThe panel meter can report values for current and voltage while in any operational mode. Steady-state current is reported to monitor voltage-clamp output. To evaluate noise, current is displayed as an rms value. The Vm setting is used under current clamp to evaluatemembrane potential. In I=0 mode, Vm is the resting membrane potential of the cell. VHOLD/IHOLD reports the setting of the Holding Command. The value isreported even when this command is switched off, allowing you to precisely set a desired holding commandin advance. The temperature of the cooled headstage circuitry may be displayed when TEMP is selected.

    Leak SubtractionIt is often convenient to subtract leak errors during an experiment. The Axopatch 200B allows you to correct leak errors as they appear without adjusting your data acquisition software settings. This is also useful when a large seal leakage is expected, as in loose-patch experiments. Leak Subtraction has been improved in the Axopatch 200B by increasing sensitivity in the resistance range most likely to be encountered by researchers.

    ZapTo go whole cell it is necessary to rupture the cell- attached patch. As an alternative to carefully controlled suction, the Axopatch 200B allows the patch to be ruptured by applying a single +1.3 V pulse for a chosen duration.

    Improved Headstage DesignThe newly engineered headstage has only one third the cross-sectional area of previous headstages, thereby greatly improving ease of access to the preparation.

    Excellent ManualWritten by scientific consultants, with the assistance of Axon Instruments staff, the Axopatch 200B manual will serve as a useful guide to the operation of the Axopatch 200B as well as an informative reference for many aspects of patch clamping.

    Pipette HoldersThe pipette holder, HL-U, is custom constructed to ensure low-noise mechanically stable recording. Two different barrel lengths are provided. HL-U accepts pipettes of 1.0 mm to 1.7 mm OD. It includes a silver wire assembly, and has a 1.0 mm OD post for suction. The Axopatch 200B is shipped with one pipette holder. For further information see HL-U Holder in the Headstages and Holders section of this catalog.