MDE GmbH has been successful for years with its workstation specially designed for the standard microelectrode technique. This was further developed for measurement of the summed voltage response (AP) of ion channels of the embryonic and adult zebrafish heart.
The workstation consists of the following units:
- Mechanical unit,
- Measuring system:
- High input impedance, low noise amplifier (EXP-INT-1),
- Data display, storage and analysis hardware/software (SPEL INTRASYS).
The mechanical unit
Its base is a highly stable, vibration-free bench, which has all the glass technical elements necessary to keep the heart preparation from adult and embryonic specimen.
Each element and its functions is presented in the following figures.
Operation modes of the organ bath for adult fish heart preparation
The physiological solution constantly flows in the organ chamber and the maintenance of the proper fluid level is provided by pump suction.
The flow of the physiological solution is suspended: tap 1 closed, tap 3 open, pump stopped. The solution is added to the organ.
The setup of equipment necessary for the experiment
One of the advantages of the mechanical design of the system is that the equipment necessary for the experiment (pl.: manipulators, organ bath, lighting, etc.) can be arranged on the top plate of the bench in the most convenient position. Another advantage is that the organ bath can be changed in the slot of the table to fit any species. Setting the position of impalement supported by the longitudinal adjustability of the organ bath. This solution allows the simultaneous measurement of multiple embryos.
The measuring system
The measuring system consists of three main units:
- EXP-INT-1 high input impedance(1012Ώ), low noise, low capacity (25pF) amplifier,
- EXP-ST-A1 isolated stimulator end stage (10V/10mA),
- SPEL INTRASYS measurement, analysis and data storage hardware-software system with stimulator module.
In order to avoid external noise contamination (since the system does not contain any filter) and to keep capacity low, a shielding ground point common with the mechanics has been designed. Through this point we connect to the shielding ground of the Faraday cage.
The EXP-INT-1 amplifier was developed for the standard microelectrode technique. The applied technical solutions allow an accurate transfer of the shape of the AP signals using glass electrodes between 10MΩ and 100MΩ. The amplifier consists of two units. One is the "INTE" high input impedance preamplifier, which can be directly connected to the electrode sleeve (e.g.: WPI, Harvard, Clark, etc.). The other element is the power amplifier, which contains the previously described circuit elements, and provides the connection to the data imaging, -storage and -analysis hardware-software system (SPEL INTRASYS).
In order to achieve these favorable parameters, the linear amplifier solution was chosen with reference earth. All the services, provided by the amplifier (e.g.: capacity compensation, resistance measurement, DC electrode contact potential cancellation etc.) can be controlled through the constant resistance (1Ω) reference earth.
SPEL INTRASYS hardware-software
The SPEL INTRASYS is a one-channel storage oscilloscope software. The software can be used to display, store and analyze extra- and intracellular potentials, triggered in the heart or any other anatomical region (e.g.: brain, bone marrow, etc.) in-vivo or in-vitro way.
Heart single-cell registration
For heart AP curves measured with standard microelectrode technique, in online mode the following parameters are displayed, analyzed and stored, during the measurement. Later, in offline mode the primer curves and the saved parameters can be reanalyzed and modified:
- RP – rest potential
- TOP – taking off potential
- APA1 – AP to the RP = OS – RP
- APA2 – AP to the MDP =OS – MDP
- MDP – minimum potential
- OS – overshot
- APD10 –10% coming back
- APD25 –25% coming back
- APD50 –50% coming back
- APD75 –75% coming back
- APD90 - 90% coming back
- APD50mV –50mV coming back
- CT – leading time
- dVmax – the fastest point of the rising branch of the sign
- dVmin – the fastest point of the descending branch of the signal
provides an opportunity to display a primary curve, stored at a specific moment, any time during the measurement, and to compare this curve with the curves measured or monitored at that moment. In addition, there is a possibility to calculate an average from the curves.
Internal stimulator with EXP-ST-A1 isolated power output stage
- Pulse Period (PP) 500 µs ... 999 s
- Delay (DE) 0.1 µs ... 999 s
- Impulse Width (PW) 10 µs ... 999 s
- Train Delay (TD) 10 µs ... 999 s
- Train Number (TN) 1 ... 999
- Max. output voltage ± 10 V, ± 100 V
- Current limit ± 10 mA, ± 100 mA
Built up from units (according to individual demand)
Upon request, the system can be set up according to individual needs from the following units:
How can it help?
The advantage of the standard microelectrode method
The recent decades proved that zebrafish is a preferred research subject in cardiac research, especially in heart physiological and pharmacological tests. Electrophysiology of the zebrafish heart shows similarity in many points to that of mammals used at present as experimental moddels (e.g.: ion channels, ECG, etc.), and it has a unique development physiological applicability. Based on the similarities revealed, the cellular techniques (standard microelectrode, patch clamp) applied for mammals also appeared in zebrafish studies.
Representative shapes of atrial (left column) and ventricular (right column) cardiac action potentials in the adult zebrafish (A), human (B) and mouse (C). APs were recorded from spontaneously beating intact zebrafish hearts at 28ºC, while human and mouse cardiac tissues were stimulated at a frequency of 1 Hz and measured at 37ºC. Zebrafish recordings were obtained from the apical half of the ventricle and from the central area of the atrium.
The advantage of standard microelectrode technique is that the transmembrane changes are recorded from the preparation impaled with the electrode through the amplifier in such a way that, in one cell of the preparation, the tip of the electrode detects electrical potential changes of the intracellular space related to the extracellular space. This technique enables the measurement of voltage changes between the extra- and intracellular space, caused by all active ion channels of the given cell. The summed signal, obtained by the measurement method, defines the formation of the curves of ECG, a heart physiological examination method used widely in the clinical practice.
With this technique it becomes possible to measure the voltage triggered by all the ion channels in the given cell, between the extra-/intracellular space. The sum signal, which is mapped by the measurement method, defines the formation of the heart physiological measurement method, the ECG curves, which are well spread in the clinical practice.
The introduction of the measuring unit
The parameters of the analogue amplifier of the standard microelectrode measuring unit described above, high input resistance and low capacity (1012 Ω/25pF) guarantee the form keeping transmission of AP signals in case of 10-90 MΩ electrode resistance. The advantageous input parameters and the asymmetrical arrangement result in low leakage current, which is – in contrast to the clamp amplifiers – ensures the proper rising slope of the AP signal, so the physiological information represented by the dV/dt value will not be distorted.
With the background support of the amplifier (EXP-INT-1) we can ensure that transmission of the AP signal and the measurement process are as undistorted as possible. In addition to that, the user is continuously informed of the technical status of the devices, ensuring an objective monitoring of the status of the biological preparation. These services are integrated into the amplifier and can be monitored via the 3 digit LED display and audio output:
- Electrode resistance measurement,
- Audio monitor.
In the measurement of individual physical features, our aim was to check not only the technical parameters of the amplifier, but also the status of the biological preparation to be measured. For this purpose, a 1Ω reference earth (REF) was constructed which is independent of the electronic and the shielding ground (GND).
As a result of the solution the capacity of the system reduces, the fluctuation of the baseline (zero level) can be stabilized and it only depends on the status of the Ag/AgCl reference electrode, which can be checked this way.
Electrode resistance measurement
The resistance is measured by the principle of conductivity measurement, by equalizing two facing, opposite-phase square waves. The two opposite-phase waves pass through the electrode and the preparation, and after equalizing, the value can be read on the LED display (measurement range: 10-100MΩ).
Baseline setting (bucking)
Measurement accuracy is deteriorated if the baseline level of the amplifier cannot be set correctly. This voltage shift can be caused by two factors:
- Incorrect setting of the amplifier offset voltage, which can be corrected by setting the offset voltages of the circuits in the amplifier.
- Contact potential at the connection point of the electrode and preparation. This problem has two possible solutions:
- Setting zero level within the amplifier (due to their construction, clamp amplifiers, e.g., use this solution),
- Deleting the potential directly at the connection point of the electrode and the preparation. This has the advantage that the potential, kept separate from the measured signal, cannot reduce the useful signal, and will not saturate the input stage. This way, the whole measuring range can be exploited, and the AP curve transmission will not be distorted. This is achieved by the ±100mV voltage level, which can be led though the REF earth, and can be set by the bucking potentiometer.
You find details by clicking on the product code.
ZFS-03 CellFish intracellular single heart cell measuring system
|EXP-INT-1||One-channel intracellular amplifier (1012Ώ) with resistance meter and capacitive compensation|
|EXP-ST-A1||Isolated stimulator with high performance power (0-30V/50mA)|
|EXP-SG-1||DC bridge amplifier (1-channel)|
|EXP-TH-1||Temperature meter amplifier (1-channel) with sensor|
|TSZ-01||Vibration-free workstation with organ bath, buffer reservoir or pump|
|Three-plane 1μm precision manipulator with magnetic base|
|SEN-12-12||Ball-mounted manipulator (360° movement, self-righting) with platinum stimulating electrode|
|CWB-02||Circulating water bath (20 litre, 20 l/min delivery speed, ±0,1°C)|
|Faraday cage (30x80x120)|
|SOFT-04||MDE - SPEL INTRASYS measuring, analysing, storing hardware/software system with stimulator module|
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