Specifications

Description of Controls

The Six-Channel Voltage Current Clamp is divided physically and functionally into sections. The upper part of the instrument contains a bank of 2, 6, or 8 (MC2, MC6, MC8) independent voltage-current clamps while the lower part (center panel on the VCC MC2) contains the pulse generator and dc clamp level controls, the Master Control, and the power switch. Controls for the independent voltage-current clamp sections will be described first. Switch positions are indicated by light emitting diode (LED) indicators.

Clamping Circuitry

The Offset is used to compensate for voltage differences between the measuring electrodes. The range is ±10 mV or ±100 mV, set by an internal jumper. Adjustment is by means of a 10-turn calibrated potentiometer knob after pressing the OFFSET switch to a + or - polarity. For electrodes that are well matched, a range of ±10 mV is sufficient. For electrodes that are not well matched, but are stable, the ±100 mV range should be selected (see Table II). Electrodes that are not stable with time should be reconditioned or discarded.

The Fluid Resistance Compensation (FRC) circuitry is used to correct for non-tissue related resistance in the chamber. If this is not done properly, measurements of tissue resistance will not be accurate. The FRC range is typically 0-100 ohms, but is also user selectable by internal jumpers (see Table II). FRC is adjusted by pressing pushbutton switch labeled PUSH TO ADJ to pass a test current then turning the 10-turn calibrated potentiometer knob to nullify the voltage change. To make the adjustment the FUNCTION switch (see below) must be in the OPEN position.

The FUNCTION switch determines whether: 1) the inputs of the voltage sensing amplifiers are to be internally grounded (ZERO, no lights), 2) the feedback circuitry is open circuited (OPEN, green light) so that the epithelium is in the "open circuit" condition, or 3) the feedback loop is completed (CLAMP, red light) so that the tissue is either voltage clamped or current clamped at the level given by the command signal (see below).

Current / Voltage - Selects whether the transepithelial current (red) or the transepithelial voltage (green) is to be clamped when the FUNCTION switch is set to CLAMP. Remote - Determines whether the selection of clamp FUNCTION, CURRENT/VOLTAGE MODE, and the clamp level are to be controlled using the front panel controls (local mode) or by external device such as a computer (REMOTE - amber LED on).

Selects either the transepithelial current (red) or voltage (green) to be displayed on the panel meter. Displayed voltage is V2-V1. Transepithelial current displays as positive for cation flow across the tissue from side 2 to side 1. For example, for the toad urinary bladder that actively absorbs the cation sodium, the transepithelial voltage is typically negative in the lumen with respect to the blood side (e.g., -50 mV). If V1 and I1 are connected to the blood side of the epithelium, then the displayed spontaneous transepithelial voltage V2-V1 will be - 50.0 mV while the short-circuit current will be positive. The meter may be read directly in mV or uA over the range ±199.9 mV or uA. Note: the gain of the current measuring amplifier may be altered using internal jumpers (see Internal Jumper Selection below).

General Operating Instructions

Start UP

Before powering up the instrument please verify the main voltage setting (see Setting the Mains Voltage ). Turn the POWER switch to ON. Allow the instrument to warm up for at least 2 minutes. Upon power up the following conditions will be established: FUNCTION switch set to ZERO (both LED's off); the MODE switches to LOCAL (REM LED off), VOLTAGE (green); METER set to display voltage (green); OFFSET set to off (both LED's off); PULSE GENERATOR set to off, bipolar, and x1; DC CLAMP LEVEL set to off; and, MASTER CONTROL off. All calibrated potentiometers should be set to 0.0 (i.e., turned fully counterclockwise).

Connect the voltage sensing and current passing electrodes to the instrument either by way of the SINGLE CHANNEL INPUT MODULES (DM_MC6 -see page 23) or via the ELECTRODE PANEL (EP6, for MC6 only).

Typical Experiment

Fill an "Ussing" type chamber with physiological saline and connect appropriate electrodes to inputs V1 and V2 for measuring of the transepithelial voltage. Connect current passing electrodes to I1 and I2 ensuring that V1 and I1 are connected to the same side of the epithelium. The digital panel meter should read zero when the METER switch is in either the VOLTAGE or CURRENT position. Switch the FUNCTION switch to OPEN (green) and the METER switch to VOLTAGE (green). The voltage (V2 -V1) is the asymmetry voltage between the voltage measuring electrodes and is the sum of asymmetry in the electrodes themselves and all liquid junction potentials in the voltage measuring circuit. To compensate for these place the OFFSET switch in either the plus (red) or minus (green) position and adjust the OFFSET potentiometer so that the voltage reads 0.0 on the panel meter. If the reading on the meter is not stable, is off scale (i.e., > ±199.9 mV), or cannot be compensated, the problem usually involves an air bubble or discontinuity in one of the voltage sensing limbs of the circuit (e.g., agar bridge or electrode). When the electrode OFFSET is properly adjusted place the METER switch on CURRENT and press and hold the FLUID RESISTANCE COMPENSATION test button labeled PUSH TO ADJUST. Verify that a current of approximately 60-68 uA is registered on the meter.

NOTE: Values less than this indicate that the resistance of the current passing bridges or electrodes is excessive. While this in itself will not prevent the clamp from working, it does limit the amount of current that can be passed and is poor experimental design in that it increases errors due to non-ideal rejection of the common mode voltage resulting from current flow between the chamber and the I1 input. It is recommended, therefore, that the resistance of the current passing bridges be kept low by making them as short as possible and/or by increasing their cross-sectional area. For reference, typical electrode resistances in the EasyMount Ussing chambers are 1-2 kΩ.

While still passing current (by continuing to press the PUSH TO ADJUST button), press the METER switch until VOLTAGE is displayed and then adjust the FLUID RES COMP potentiometer so that V2-V1 on the panel meter again reads 0.0. At this point the value of the resistance due to the fluid in series with the tissue may be read from the calibrated dial and will be compensated for automatically at any current - i.e., the actual voltage across the epithelium will be determined and will appear on the digital panel meter and at the VOLTAGE OUTPUT BNC connector (at x10 gain). In the event that a current cannot be passed when the test button is pushed, the problem usually involves an air bubble in one of the current passing agar bridges.

Now place the FUNCTION switch back on ZERO (both LED's off) and mount a tissue in the chamber being careful not to change the distance between the tips of the voltage sensing electrodes. Place the FUNCTION switch on OPEN and the METER switch on VOLTAGE to read the open circuit voltage generated by the tissue (already corrected for electrode asymmetry potentials). Place the FUNCTION switch on CLAMP with the MODE switch on VOLTAGE and verify that (V2-V1) now reads 0.0 - i.e., the tissue is short-circuited. Place the METER switch to CURRENT to read the short circuit current, Isc. The transepithelial voltage may now be clamped to a user specified voltage using the CLAMP VOLTAGE or CURRENT controls and/or the PULSE GENERATOR circuitry.

The MASTER CONTROL section will permit you change clamp function, clamp modes and meter settings for all clamp channels. Because all installed clamp channels will follow commands from the MASTER CONTROL, using this feature requires that each installed channel must either be connected to a chamber via electrodes or be connected to a dummy load. For instruments shipped with the DM_MC6 input stage, a dummy test membrane is built into the headstage so that the slide switch simply needs to be pushed to "Test" instead of "Operate" (see instructions DM_MC6 use - p. 16). For instruments shipped with the 6-channel electrode input panel, a separate modular plug is provided for each clamp channel which serves as a dummy load and should be connected via the SINGLE CHANNEL INPUT MODULE connector for each unused clamp channel. The reason for this is that if no load is connected to an individual clamp channel, then placing that channel in voltage clamp mode will cause the output of the clamp amplifier to saturate and may influence the output of other clamp channels (via the shared power supply) resulting in erroneous measurements. This is circumvented either by not using MASTER CONTROL when all channels are not in use, or by using the dummy loads on unused channels.

Remote Control When the MODE switch is placed on REMOTE the front panel FUNCTION (OPEN or CLAMP) and MODE (clamp I or V) controls are disabled. All other controls are unaffected. Control of these disabled features must be supplied via the REMOTE interface on the rear panel. Details of this feature are given below.

Remote Interface Connections On the rear panel is a 25 pin female D sub-miniature connector by which interface may be made to an external controller such as a computer or a simple battery and switch. A compatible interface cable is provided with the ACQUIRE & ANALYZE hardware/software data acquisition system.

The pin connections are shown in Table I.

For more information, please see our Manual